FoxO Transcription Factors Research Articles

Developmental history modulates adult olfactory behavioral preferences via regulation of chemoreceptor expression in Caenorhabditiselegans.

Developmental experiences play critical roles in shaping adult physiology and behavior. We and others previously showed that adult Caenorhabditiselegans which transiently experienced dauer arrest during development (postdauer) exhibit distinct gene expression profiles as compared to control adults which bypassed the dauer stage. In particular, the expression patterns of subsets of chemoreceptor genes are markedly altered in postdauer adults. Whether altered chemoreceptor levels drive behavioral plasticity in postdauer adults is unknown. Here, we show that postdauer adults exhibit enhanced attraction to a panel of food-related attractive volatile odorants including the bacterially produced chemical diacetyl. Diacetyl-evoked responses in the AWA olfactory neuron pair are increased in both dauer larvae and postdauer adults, and we find that these increased responses are correlated with upregulation of the diacetyl receptor ODR-10 in AWA likely via both transcriptional and posttranscriptional mechanisms. We show that transcriptional upregulation of odr-10 expression in dauer larvae is in part mediated by the DAF-16 FOXO transcription factor. Via transcriptional profiling of sorted populations of AWA neurons from control and postdauer animals, we further show that the expression of a subset of additional chemoreceptor genes in AWA is regulated similarly to odr-10 in postdauer animals. Our results suggest that developmental experiences may be encoded at the level of olfactory receptor regulation, and provide a simple mechanism by which C. elegans is able to precisely modulate its behavioral preferences as a function of its current and past experiences.

FOXO1 differentially regulates bone formation in young and aged mice

It is a great challenge to develop a safe and effective treatment strategy for age-related osteoporosis and fracture healing. As one of the four FOXO transcription factors, FOXO1 is essential for cell proliferation, survival, senescence, energy metabolism, and oxidative stress in various cells. Our previous study demonstrated that specific Foxo1 gene deletion in osteoblasts in young mice results in bone loss while that in aged mice shows the opposite effect. However, the mechanism underlying the differential regulation of bone metabolism by FOXO1 remains to be elucidated. In this study, we generated osteoblast-specific Foxo1 knockout mice by using Foxo1fl/fl and Bglap-Cre mice. In young mice, Foxo1 gene deletion inhibits osteoblast differentiation, leading to a decreased osteoblast number and decreased bone formation rate because of the weakened ability to resist oxidative stress, eventually resulting in bone loss and delayed healing of bone defects. In aged mice, high levels of reactive oxygen species (ROS) promote the diversion of CTNNB1 (β-catenin) from T cell factor 4 (TCF4)- to FOXO1-mediated transcription, thereby inhibiting Wnt/β-catenin signaling and leading to decreased osteogenic activity. Conversely, FOXO1 deficiency indirectly promotes the binding of β-catenin and TCF4 and activates Wnt/β-catenin signaling, thereby alleviating age-related bone loss and improving bone defect healing. Our study proves that FOXO1 has differential effects on bone metabolism in young and aged mice and elucidates its underlying mechanism. Further, this study provides a new perspective on the treatment of age-related osteoporosis.

Perspectives for Forkhead box transcription factors in diabetic cardiomyopathy: Their therapeutic potential and possible effects of salvianolic acids.

Diabetic cardiomyopathy (DCM) is the primary cause of morbidity and mortality in diabetic cardiovascular complications, which initially manifests as cardiac hypertrophy, myocardial fibrosis, dysfunctional remodeling, and diastolic dysfunction, followed by systolic dysfunction, and eventually end with acute heart failure. Molecular mechanisms underlying these pathological changes in diabetic hearts are complicated and multifactorial, including but not limited to insulin resistance, oxidative stress, lipotoxicity, cardiomyocytes apoptosis or autophagy, inflammatory response, and myocardial metabolic dysfunction. With the development of molecular biology technology, accumulating evidence illustrates that members of the class O of Forkhead box (FoxO) transcription factors are vital for maintaining cardiomyocyte metabolism and cell survival, and the functions of the FoxO family proteins can be modulated by a wide variety of post-translational modifications including phosphorylation, acetylation, ubiquitination, arginine methylation, and O-glycosylation. In this review, we highlight and summarize the most recent advances in two members of the FoxO family (predominately FoxO1 and FoxO3a) that are abundantly expressed in cardiac tissue and whose levels of gene and protein expressions change as DCM progresses, with the goal of providing valuable insights into the pathogenesis of diabetic cardiovascular complications and discussing their therapeutic potential and possible effects of salvianolic acids, a natural product.

Potential roles of FoxO in promoting longevity in larger Argopecten scallops

Bay scallops are commercially important bivalve species for fisheries and aquaculture in China, the USA, Peru and Chile, but their small size, short lifespan (<14 months) and serious inbreeding depression restrict the sustainable development of their aquaculture. Compared with bay scallops, some interspecific hybrids of bay scallops and Peruvian scallops exhibit longer lifespans and significantly larger sizes, which may result from the longevity genes of Peruvian scallops, which have lifespans of 7–10 years. The FoxO transcription factor integrates signals from different longevity-related pathways that play a crucial role in aging and longevity in terrestrial model organisms. However, limited information is available on its roles in the longevity of marine animals. Here, we extended this paradigm using Peruvian scallops and bay scallops, which have distinct lifespans. Cloning and sequence analysis of the FoxO ORFs (ApFoxO and AiFoxO) revealed 22 synonymous, 7 nonsynonymous SNPs and two InDels between the two species. The amino acid variations (Q27 and E28) near the AKT phosphorylation site (T21) may affect the activities of ApFoxO for longevity. Nutrient restriction could extend lifespan in terrestrial model organisms, and the expression of ApFoxO and AiFoxO both significantly increased under nutrient restriction, but the response in bay scallops was faster and more robust than that in Peruvian scallops. Furthermore, ionizing radiation caused significantly lower mortality and higher FoxO expression in Peruvian scallops. These results suggested that FoxO may play a protective role in longevity through nutrient availability and DNA repair for genomic stability. The significantly decreased expression of CAT and Mn-SOD and the increased β-GAL activity (a marker for senescence) after FoxO downregulation suggested that FoxO may contribute to longevity by controlling ROS with its antioxidase activity. The results of our work provide the basis for understanding the role of FoxO in longevity in scallops and its potential as a biomarker for selecting larger hybrids to benefit scallop aquaculture.

Integrative Analysis Between Genome-Wide Association Study and Expression Quantitative Trait Loci Reveals Bovine Muscle Gene Expression Regulatory Polymorphisms Associated With Intramuscular Fat and Backfat Thickness.

Understanding the architecture of gene expression is fundamental to unravel the molecular mechanisms regulating complex traits in bovine, such as intramuscular fat content (IMF) and backfat thickness (BFT). These traits are economically important for the beef industry since they affect carcass and meat quality. Our main goal was to identify gene expression regulatory polymorphisms within genomic regions (QTL) associated with IMF and BFT in Nellore cattle. For that, we used RNA-Seq data from 193 Nellore steers to perform SNP calling analysis. Then, we combined the RNA-Seq SNP and a high-density SNP panel to obtain a new dataset for further genome-wide association analysis (GWAS), totaling 534,928 SNPs. GWAS was performed using the Bayes B model. Twenty-one relevant QTL were associated with our target traits. The expression quantitative trait loci (eQTL) analysis was performed using Matrix eQTL with the complete SNP dataset and 12,991 genes, revealing a total of 71,033 cis and 36,497 trans-eQTL (FDR < 0.05). Intersecting with QTL for IMF, we found 231 eQTL regulating the expression levels of 117 genes. Within those eQTL, three predicted deleterious SNPs were identified. We also identified 109 eQTL associated with BFT and affecting the expression of 54 genes. This study revealed genomic regions and regulatory SNPs associated with fat deposition in Nellore cattle. We highlight the transcription factors FOXP4, FOXO3, ZSCAN2, and EBF4, involved in lipid metabolism-related pathways. These results helped us to improve our knowledge about the genetic architecture behind important traits in cattle.

The Rab GTPase activating protein TBC-2 regulates endosomal localization of DAF-16 FOXO and lifespan.

FOXO transcription factors have been shown to regulate longevity in model organisms and are associated with longevity in humans. To gain insight into how FOXO functions to increase lifespan, we examined the subcellular localization of DAF-16 in C. elegans. We show that DAF-16 is localized to endosomes and that this endosomal localization is increased by the insulin-IGF signaling (IIS) pathway. Endosomal localization of DAF-16 is modulated by endosomal trafficking proteins. Disruption of the Rab GTPase activating protein TBC-2 increases endosomal localization of DAF-16, while inhibition of TBC-2 targets, RAB-5 or RAB-7 GTPases, decreases endosomal localization of DAF-16. Importantly, the amount of DAF-16 that is localized to endosomes has functional consequences as increasing endosomal localization through mutations in tbc-2 reduced the lifespan of long-lived daf-2 IGFR mutants, depleted their fat stores, and DAF-16 target gene expression. Overall, this work identifies endosomal localization as a mechanism regulating DAF-16 FOXO, which is important for its functions in metabolism and aging.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

Maternal and perinatal obesity induce bronchial obstruction and pulmonary hypertension via IL-6-FoxO1-axis in later life

Obesity is a pre-disposing condition for chronic obstructive pulmonary disease, asthma, and pulmonary arterial hypertension. Accumulating evidence suggests that metabolic influences during development can determine chronic lung diseases (CLD). We demonstrate that maternal obesity causes early metabolic disorder in the offspring. Here, interleukin-6 induced bronchial and microvascular smooth muscle cell (SMC) hyperproliferation and increased airway and pulmonary vascular resistance. The key anti-proliferative transcription factor FoxO1 was inactivated via nuclear exclusion. These findings were confirmed using primary SMC treated with interleukin-6 and pharmacological FoxO1 inhibition as well as genetic FoxO1 ablation and constitutive activation. In vivo, we reproduced the structural and functional alterations in offspring of obese dams via the SMC-specific ablation of FoxO1. The reconstitution of FoxO1 using IL-6-deficient mice and pharmacological treatment did not protect against metabolic disorder but prevented SMC hyperproliferation. In human observational studies, childhood obesity was associated with reduced forced expiratory volume in 1 s/forced vital capacity ratio Z-score (used as proxy for lung function) and asthma. We conclude that the interleukin-6-FoxO1 pathway in SMC is a molecular mechanism by which perinatal obesity programs the bronchial and vascular structure and function, thereby driving CLD development. Thus, FoxO1 reconstitution provides a potential therapeutic option for preventing this metabolic programming of CLD.

A case report of Apert syndrome with review of literatures

Apert syndrome is a rare syndrome which presents with craniosynostosis, severe syndactyly, and dysmorphic facial features. It is mainly caused by a new mutation in fibroblast growth factor receptor-2 gene. Up-regulation of this gene results in bone fusion and nuclear deficiency of the transcription factor FoxO1 which is a key transcription factor in the pathogenesis of acne. We present herein a 19-year-old man with nodulocystic acne associated with acrocephaly, prominent forehead, ocular hypertelorism, short and broad nose, high arched palate, maxillary hypoplasia, dental crowding and ectopia, severe bilateral syndactyly of the hands and feet. Apert syndrome was diagnosed for him based on mentioned clinical findings. Isotretinoin 20 mg/day was prescribed for nodulocystic acne with significant improvement two months later. Severe acne in early puberty associated with synostosis is the hallmark of Apert syndrome and we should mindful of these syndromic cases in dermatology clinic.

The Parkinson's disease variant rs356182 regulates neuronal differentiation independently from alpha-synuclein.

One of the most significant risk variants for Parkinson's disease (PD), rs356182, is located at the PD-associated locus near the alpha-synuclein (α-syn) encoding gene, SNCA. SNCA-proximal variants, including rs356182, are thought to function in PD risk through enhancers via allele-specific regulatory effects on SNCA expression. However, this interpretation discounts the complex activity of genetic enhancers and possible non-conical functions of α-syn. Here we investigated a novel risk mechanism for rs356182. We use CRISPR-Cas9 in LUHMES cells, a model for dopaminergic midbrain neurons, to generate precise hemizygous lesions at rs356182. The PD-protective (A/-), PD-risk (G/-) and wild-type (A/G) clones were neuronally differentiated and then compared transcriptionally and morphologically. Among the affected genes was SNCA, whose expression was promoted by the PD-protective allele (A) and repressed in its absence. In addition to SNCA, hundreds of genes were differentially expressed and associated with neurogenesis and axonogenesis-an effect not typically ascribed to α-syn. We also found that the transcription factor FOXO3 specifically binds to the rs356182 A-allele in differentiated LUHMES cells. Finally, we compared the results from the rs356182-edited cells to our previously published knockouts of SNCA and found only minimal overlap between the sets of significant differentially expressed genes. Together, the data implicate a risk mechanism for rs356182 in which the risk-allele (G) is associated with abnormal neuron development, independent of SNCA expression. We speculate that these pathological effects manifest as a diminished population of dopaminergic neurons during development leading to the predisposition for PD later in life.

Hepatic p53 is regulated by transcription factor FOXO1 and acutely controls glycogen homeostasis

The tumor suppressor p53 is involved in the adaptation of hepatic metabolism to nutrient availability. Acute deletion of p53 in the mouse liver affects hepatic glucose and triglyceride metabolism. However, long-term adaptations upon the loss of hepatic p53 and its transcriptional regulators are unknown. Here we show that short-term, but not chronic, liver-specific deletion of p53 in mice reduces liver glycogen levels, and we implicate the transcription factor forkhead box O1 protein (FOXO1) in the regulation of p53 and its target genes. We demonstrate that acute p53 deletion prevents glycogen accumulation upon refeeding, whereas a chronic loss of p53 associates with a compensational activation of the glycogen synthesis pathway. Moreover, we identify fasting-activated FOXO1 as a repressor of p53 transcription in hepatocytes. We show that this repression is relieved by inactivation of FOXO1 by insulin, which likely mediates the upregulation of p53 expression upon refeeding. Strikingly, we find that high-fat diet–induced insulin resistance with persistent FOXO1 activation not only blunted the regulation of p53 but also the induction of p53 target genes like p21 during fasting, indicating overlapping effects of both FOXO1 and p53 on target gene expression in a context-dependent manner. Thus, we conclude that p53 acutely controls glycogen storage in the liver and is linked to insulin signaling via FOXO1, which has important implications for our understanding of the hepatic adaptation to nutrient availability.

Cholinergic Senescence in the Ts65Dn Mouse Model for Down Syndrome

Down syndrome (DS) induces a variable phenotype including intellectual disabilities and early development of Alzheimer’s disease (AD). Moreover, individuals with DS display accelerated aging that affects diverse organs, among them the brain. The Ts65Dn mouse is the most widely used model to study DS. Progressive loss of cholinergic neurons is one of the hallmarks of AD present in DS and in the Ts65Dn model. In this study, we quantify the number of cholinergic neurons in control and Ts65Dn mice, observing a general reduction in their number with age but in particular, a greater loss in old Ts65Dn mice. Increased expression of the m1 muscarinic receptor in the hippocampus counteracts this loss. Cholinergic neurons in the Ts65Dn mice display overexpression of the early expression gene c-fos and an increase in the expression of β-galactosidase, a marker of senescence. A possible mechanism for senescence induction could be phosphorylation of the transcription factor FOXO1 and its retention in the cytoplasm, which we are able to confirm in the Ts65Dn model. In our study, using Ts65Dn mice, we observe increased cholinergic activity, which induces a process of early senescence that culminates in the loss of these neurons.

FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes

Upon injury, the liver is capable of substantial regeneration from the original tissue until an appropriate functional size. The underlying mechanisms controlling the liver regeneration processes are not well elucidated. Previous studies have proposed that the transcription factor FoxO3 is involved in various liver diseases, but its exact role in the regulation of liver regeneration remains largely unclear. To directly test the detailed role of FoxO3 in liver regeneration, both a constitutive Albumin-Cre driver line and adeno-associated virus serotype 8 (AAV8)-Tbg-Cre (AAV-Cre)-injected adult FoxO3fl/fl mice were subjected to 70% partial hepatectomy (PH). Our data demonstrate that FoxO3 deletion accelerates liver regeneration primarily by limiting polyploidization and promoting the proliferation of hepatocytes during liver regeneration. RNA-seq analysis indicates that FoxO3 deficiency greatly alters the expression of gene sets associated with cell proliferation and apoptosis during liver regeneration. Chromatin immunoprecipitation-PCR (ChIP-PCR) and luciferase reporter assays reveal that FoxO3 promotes the expression of Nox4 but suppresses the expression of Nr4a1 in hepatocytes. AAV8 virus-mediated overexpression of Nox4 and knockdown of Nr4a1 significantly suppressed hepatocyte proliferation and liver regeneration in FoxO3-deficient mice. We demonstrate that FoxO3 negatively controls hepatocyte proliferation through Nox4 upregulation and Nr4a1 downregulation, thereby ensuring appropriate functional regeneration of the liver. Our findings provide novel mechanistic insight into the therapeutic mechanisms of FoxO3 in liver damage and repair.

Phosphoproteomic studies of alamandine signaling in CHO-MrgD and human pancreatic carcinoma cells: An antiproliferative effect is unveiled.

Alamandine is a heptapeptide from the renin-angiotensin system (RAS) with similar structure/function to angiotensin-(1-7) [ang-(1-7)], but they act via different receptors. It remains elusive whether alamandine is an antiproliferative agent like ang-(1-7). The goal of this study was to evaluate the potential antiproliferative activity of alamandine and the underlying cellular signaling. We evaluated alamandine effect in the tumoral cell lines Mia PaCa-2 and A549, and in the nontumoral cell lines HaCaT, CHO and CHO transfected with the alamandine receptor MrgD (CHO-MrgD). Alamandine was able to reduce the proliferation of the tumoral cell lines in a MrgD-dependent fashion. We did not observe any effect in the nontumoral cell lines tested. We also performed proteomics and phosphoproteomics to study the alamandine signaling in Mia PaCa-2 and CHO-MrgD. Data suggest that alamandine induces a shift from anaerobic to aerobic metabolism in the tumoral cells, induces a negative regulation of PI3K/AKT/mTOR pathway and activates the transcriptional factor FoxO1; events that could explain, at least partially, the observed antiproliferative effect of alamandine. This study provides for the first time a comprehensive investigation of the alamandine signaling in tumoral (Mia PaCa-2) and nontumoral (CHO-MrgD) cells, highlighting the antiproliferative activity of alamandine/MrgD and its possible antitumoral effect.

1335-P: Deciphering Liver Glycome Regulation by the PGC-1/FN3K Axis

Diabetes in general, and specifically in the context of obesity, is characterized by hyperglycemia resulting in non-enzymatic glycation of proteins, a key event in the pathophysiology of diabetes and its chronic complications. Yet, the full scope of molecular targets for glycation, particularly in liver, is incompletely understood. De-glycation, which removes the attached sugars, is controlled intracellularly by fructosamine-3-kinase (FN3K) , a kinase that phosphorylates the attached sugar and has been proposed to have a protective role against the deleterious effects of glycation related to diabetes. However, FN3K has been suggested to be ubiquitously expressed and to act as a constitutive housekeeping gene. Furthermore, whether intracellular de-glycation is regulated in physiological contexts and what factors are involved in its regulation remain unknown. Our data identify that regulation of protein glycation in liver is controlled by the key metabolic regulators PGC-1s (PGC-1alpha and PGC-1beta) in response to metabolic cues, particularly in the fed state. Liver-specific deletion of PGC-1s results in reduction of Fn3k expression and concomitant increase in specific protein glycation. In accordance, overexpression of PGC-1alpha in liver-derived cells induces Fn3k and a reduction in protein glycation. Purification of glycated proteins followed by mass spectrometric analysis reveals significant alterations in intracellular protein glycation in response to PGC1-alpha expression. Mechanistically, PGC-1alpha effect on Fn3k involves the activity of the transcription factor Foxo1. Thus, in liver, fasting and re-feeding govern intracellular protein glycation via PGC-1 dependent induction of FN3K. Our work reveals the scope and dynamic nature of the liver glycome, establishing the PGC1/FN3K axis as a key regulator of protein glycation. Disclosure E.Glick saar: None. Y.G.Daime: None. A.Tirosh: Advisory Panel; Abbott Diagnostics, AstraZeneca, Boehringer Ingelheim International GmbH, Merck &amp; Co., Inc., Novo Nordisk, Sanofi, Consultant; Bayer AG, DreaMed Diabetes, Ltd., Research Support; Medtronic, Speaker’s Bureau; Eli Lilly and Company. N.Minsky: None.

Plasma miR-193b-3p Is Elevated in Type 2 Diabetes and Could Impair Glucose Metabolism.

ObjectiveTo explore differentially expressed miRNAs in type 2 diabetes and their potential cellular functions.MethodsWe screened plasma miRNAs by miRNA array analysis and validated them by TaqMan real-time PCR in 113 newly diagnosed, untreated type 2 diabetes cases and 113 healthy controls. Low-abundance plasma proteins encoded by miR-193b-3p target genes were explored in this study population. We further investigated the potential cellular functions of the differentially expressed miRNAs in HepG2 cells.ResultsmiR-193b-3p was differentially expressed in type 2 diabetes cases compared to healthy controls (fold change = 2.01, P = 0.006). Plasma levels of triosephosphate isomerase (TPI1, a protein involved in the glycolytic pathway) decreased in type 2 diabetes cases (fold change = 1.37, P = 0.002). The effect of miR-193b-3p on TPI1 was verified by transfection of miR-193b-3p into HepG2 cells. miR-193b-3p inhibited the expression of YWHAZ/14-3-3ζ in the PI3K-AKT pathway, subsequently altering the expression of FOXO1 and PCK1. After transfection, cells were incubated in glucose-free medium for another 4 h. Glucose levels in medium from cells with elevated miR-193b-3p levels were significantly higher than those in medium from negative control cells (P = 0.016). In addition, elevated miR-193b-3p reduced glucose uptake by inhibiting insulin receptor (IR) and GLUT2 expression.ConclusionPlasma miR-193b-3p levels increased in type 2 diabetes cases, and TPI1 levels decreased in both plasma and HepG2 cells with increased miR-193b-3p levels, while extracellular lactate levels did not significantly changed. Moreover, miR-193b-3p may affect glucose metabolism by directly targeting YWHAZ/14-3-3ζ and upregulating the transcription factor FOXO1 downstream of the PI3K-AKT pathway.

TRAF3IP2 regulated by FOXO4 affects fibroblast proliferation, migration, and extracellular matrix deposition in keloid through the TGF-β1/Smad pathway.

Keloids are "tumor-like" scars that grow beyond the boundary of injury. Its pathogenesis is complex. This paper will discuss the pathogenesis of keloid from the transcriptional regulation mechanism of TRAF3IP2. IL-17 was utilized to induce human keloid fibroblasts (KFs) and normal dermal fibroblasts. With the application of RT-qPCR and Western blot, TRAF3IP2 expression was detected. Subsequently, the expression of TRAF3IP2 was interfered by cell transfection and the effects of interfering TRAF3IP2 on cell proliferative rate, migration rate, and extracellular matrix were assessed with CCK-8, Wound Healing, immunofluorescence, and Western blot techniques. Proliferation, migration, and (ECM) deposition were detected by JASPAR software predicted the binding sites of transcription factors FOXO4 and TRAF3IP2 promoters. The relationship between FOXO4 and TRAF3IP2 was verified by Dual luciferase activity assay and ChIP. Finally, the expression of TRAF3IP2 and FOXO4 was interfered simultaneously to further explore the mechanism. TRAF3IP2 was enhanced in IL-17 induced KFs. Interference with TRAF3IP2 imparted suppressive effects on the proliferation, migration, and ECM deposition of KFs. FOXO4 could inhibit TRAF3IP2 transcription, and interference with FOXO4 reversed the effect of TRAF3IP2 down-regulation on KFs via TGF-β1/Smad pathway. TRAF3IP2 was regulated by FOXO4 and affected fibroblast proliferation, migration, and ECM deposition in keloid through the TGF-β1/Smad pathway.

MicroRNA-92a promotes CNS autoimmunity by modulating the regulatory and inflammatory T cell balance.

A disequilibrium between immunosuppressive Tregs and inflammatory IL-17–producing Th17 cells is a hallmark of autoimmune diseases, including multiple sclerosis (MS). However, the molecular mechanisms underlying the Treg and Th17 imbalance in CNS autoimmunity remain largely unclear. Identifying the factors that drive this imbalance is of high clinical interest. Here, we report a major disease-promoting role for microRNA-92a (miR-92a) in CNS autoimmunity. miR-92a was elevated in experimental autoimmune encephalomyelitis (EAE), and its loss attenuated EAE. Mechanistically, miR-92a mediated EAE susceptibility in a T cell–intrinsic manner by restricting Treg induction and suppressive capacity, while supporting Th17 responses, by directly repressing the transcription factor Foxo1. Although miR-92a did not directly alter Th1 differentiation, it appeared to indirectly promote Th1 cells by inhibiting Treg responses. Correspondingly, miR-92a inhibitor therapy ameliorated EAE by concomitantly boosting Treg responses and dampening inflammatory T cell responses. Analogous to our findings in mice, miR-92a was elevated in CD4+ T cells from patients with MS, and miR-92a silencing in patients’ T cells promoted Treg development but limited Th17 differentiation. Together, our results demonstrate that miR-92a drives CNS autoimmunity by sustaining the Treg/Th17 imbalance and implicate miR-92a as a potential therapeutic target for MS.

Regulation of the Age‐Dependent Activity of the FOXO Transcription Factor DAF‐16 in Adult Caenorhabditis elegans Roundworms

The insulin and insulin‐like growth factor signaling (IIS) pathway regulates stress resistance and contributes to longevity in evolutionarily diverse species. In the roundworm Caenorhabditis elegans the lifespan of daf‐2 mutants, which lack functional insulin receptors, is twice that of wildtype animals. This phenotype is entirely dependent upon the FOXO transcription factor DAF‐16. In the absence of negative regulation through the IIS pathway, DAF‐16 upregulates genes important for stress resistance, defense, and repair. While it can be activated in response to environmental insults, in young animals DAF‐16 is normally inhibited. However, we and others have demonstrated that the activity of DAF‐16 increases in an age‐dependent manner. The mechanism by which DAF‐16 is regulated during aging has not yet been defined. Here we used a reverse genetic approach in C. elegans to investigate the function of genes that act upstream in the insulin signaling pathway to ask about their potential roles in modulating DAF‐16 activity in post‐reproductive adult animals. We tested the hypothesis that activation of DAF‐2 and the ensuing activation of the PDK‐1 kinase must be blocked in order to permit DAF‐16 to regulate its transcriptional targets. By analyzing the expression of a GFP‐labeled transcriptional reporter and by measuring gene expression levels by qRT‐PCR, we found that in an age‐dependent manner DAF‐18, an ortholog of mammalian PTEN that prevents PDK‐1 from being activated, is necessary for the increased expression of DAF‐16 targets over time. Further, we found that RNAi targeting daf‐18 reduced the ability of adult but not larval stage animals to resist infection by the bacterial pathogen Pseudomonas aeruginosa. This indicates that DAF‐18 is required for DAF‐16‐mediated innate immunity later in life. Our observations raise the intriguing possibility that an agonist engages DAF‐2 to initiate signaling through the IIS pathway at the same time that DAF‐16 is activated during adulthood. Previous studies established a role for micro RNAs (miRNAs) in regulating DAF‐2 activity, and we asked whether this could be true in the context of aging by knocking down the expression of alg‐2, an argonaute protein involved in miRNA biogenesis. We reasoned that expression of a DAF‐2 ligand might be controlled post‐transcriptionally by miRNA. Our functional analysis implies a possible genetic interaction between alg‐2 and daf‐18 that is most evident in adult C. elegans, suggesting that their gene products may act in the same pathway that ultimately governs DAF‐16 function. Together, our results reveal elements of a transcriptional regulatory mechanism that is specific to adult animals and that involves evolutionarily conserved components, suggesting its general relevance to the aging process in diverse organisms.

FoxO1 Promotes Liver Fibrosis through TGF‐β1 mediated Hepatic Stellate Cell Activation

The O‐class of the forkhead transcription factor FoxO1 is a crucial factor mediating the action of insulin→PI3K→Akt and governs diverse cellular processes. To investigate the role of FoxO1 in control of liver fibrosis, we generated liver‐specific deletion of FoxO1 gene in mice (L‐F1KO). Using the mouse model, we injected intraperitoneally (i.p.) corn oil or 20% solution of carbon tetrachloride (CCL4) twice per week for 4 weeks (with 2.5 ul/g body weight) in WT and L‐F1KO mice. H&amp;E staining and Sirius Red staining revealed that CCl4‐induced liver injury and fibrosis were significantly appeared, but the effects were largely attenuated in L‐F1KO mice. Moreover, we found that both mRNA and protein levels of TGF‐β1, a cytokine that activates hepatic stellate cell (HSC) for fibrogenesis, significantly decreased in the liver L‐F1KO mice compared to WT mice in response to CCL4 treatment. In primary hepatocytes, with FoxO1‐loss of function and gain‐of function, we confirmed the regulation of TGF‐β1 expression by FoxO1. Moreover, conditional medium (CM) collected from WT hepatocytes treated with CCL4 activated human HSC cell line (LX‐2), such effect was attenuated by FoxO1 deletion in primary hepatocytes or neutralization of TGF‐β1 in the CM using TGF‐β1 antibody. These results indicate a crosstalk between hepatocytes and HSC via FoxO1‐regualted TGF‐β1. To further confirm this concept in vivo, we overexpressed FoxO1 in the liver of WT and liver‐specific TGF‐β1 deficient (L‐TGF‐β1KO) mice with adenovirus expressing FoxO1 (adFoxO1), and injected (i.p.) these mice with CCL4. We found that FoxO1 overexpression in WT mice promoted CCL4‐induced liver fibrosis and HSC activation, and such effect was blocked in L‐TGF‐β1KO mice. Taken together, our data indicate that hepatic FoxO1 promotes CCL4‐inducled liver fibrosis via upregulating TGF‐β1 expression in hepatocytes and stimulating TGF‐β1‐mediated HSC activation. Hepatic FoxO1 may be a therapeutic target for prevention and treatment of liver fibrosis.