Fam96a is essential for the host control of Toxoplasma gondii infection by fine-tuning macrophage polarization via an iron-dependent mechanism.

BACKGROUND: Toxoplasmosis affects a quarter of the world's population. Toxoplasma gondii (T.gondii) is an intracellular parasitic protozoa. Macrophages are necessary for proliferation and spread of T.gondii by regulating immunity and metabolism. Family with sequence similarity 96A (Fam96a; formally named Ciao2a) is an evolutionarily conserved protein that is highly expressed in macrophages, but whether it play a role in control of T. gondii infection is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we utilized myeloid cell-specific knockout mice to test its role in anti-T. gondii immunity. The results showed that myeloid cell-specific deletion of Fam96a led to exacerbate both acute and chronic toxoplasmosis after exposure to T. gondii. This was related to a defectively reprogrammed polarization in Fam96a-deficient macrophages inhibited the induction of immune effector molecules, including iNOS, by suppressing interferon/STAT1 signaling. Fam96a regulated macrophage polarization process was in part dependent on its ability to fine-tuning intracellular iron (Fe) homeostasis in response to inflammatory stimuli. In addition, Fam96a regulated the mitochondrial oxidative phosphorylation or related events that involved in control of T. gondii. CONCLUSIONS/SIGNIFICANCE: All these findings suggest that Fam96a ablation in macrophages disrupts iron homeostasis and inhibits immune effector molecules, which may aggravate both acute and chronic toxoplasmosis. It highlights that Fam96a may autonomously act as a critical gatekeeper of T. gondii control in macrophages.

A commensal protozoan attenuates Clostridioides difficile pathogenesis in mice via arginine-ornithine metabolism and host intestinal immune response.

Antibiotic-induced dysbiosis is a major risk factor for Clostridioides difficile infection (CDI), and fecal microbiota transplantation (FMT) is recommended for treating CDI. However, the underlying mechanisms remain unclear. Here, we show that Tritrichomonas musculis (T.mu), an integral member of the mouse gut commensal microbiota, reduces CDI-induced intestinal damage by inhibiting neutrophil recruitment and IL-1ß secretion, while promoting Th1 cell differentiation and IFN-γ secretion, which in turn enhances goblet cell production and mucin secretion to protect the intestinal mucosa. T.mu can actively metabolize arginine, not only influencing the host's arginine-ornithine metabolic pathway, but also shaping the metabolic environment for the microbial community in the host's intestinal lumen. This leads to a relatively low ornithine state in the intestinal lumen in C. difficile-infected mice. These changes modulate C. difficile's virulence and the host intestinal immune response, and thus collectively alleviating CDI. These findings strongly suggest interactions between an intestinal commensal eukaryote, a pathogenic bacterium, and the host immune system via inter-related arginine-ornithine metabolism in the regulation of pathogenesis and provide further insights for treating CDI.

Dietary ingredient change induces a transient MyD88-dependent mucosal enteric glial cell response and promotes obesity.

OBJECTIVE: Consumption of a modern Western-type high-fat low-fiber diet increases the risk of obesity. However, how a host responds to such a diet, especially during the early period of dietary transition from a previous low-fat and fiber-rich diet, remains poorly explored. METHODS: Wild-type C57BL/6 mice were fed a normal chow diet or a high-fat diet. Enteric glial cell (EGC) activation was detected through quantitative real-time PCR (qRT-PCR), immunoblotting and immunohistology analysis. Fluorocitrate or genetic deletion of glial fibrillary acidic protein (GFAP)-positive glial-intrinsic myeloid differentiation factor 88 (Myd88) was used to inhibit EGC activation, and the effect of a high-fat diet on obesity was further investigated. The role of MYD88-dependent sensing of commensal products in adipocyte was observed to analyze the effect of obesity. RESULTS: A dietary shift from a normal chow diet to a high-fat diet in mice induced a transient early-phase emergence of a GFAP-positive EGC network in the lamina propria of the ileum, accompanied with an increase in glial-derived neurotrophic factor production. Inhibition of glial cell activity blocked this response. GFAP-positive glial Myd88 knockout mice gained less body weight after high-fat diet (HFD) feeding than littermate controls. In contrast, adipocyte deletion of Myd88 in mice had no effect on weight gain but instead exacerbated glucose intolerance. Furthermore, short-term fluorocitrate intervention during HFD feeding attenuated body weight gain. CONCLUSIONS: Our findings indicate that EGCs are early responders to intestinal ecosystem changes and the GFAP-positive glial Myd88 signaling participates in regulating obesity.

FAM96A is essential for maintaining organismal energy balance and adipose tissue homeostasis in mice.

The iron (Fe) metabolism plays important role in regulating systemic metabolism and obesity development. The Fe inside cells can form iron-sulfur (Fe-S) clusters, which are usually assembled into target proteins with the help of a conserved cluster assembly machinery. Family with sequence similarity 96A (FAM96A; also designated CIAO2A) is a cytosolic Fe-S assembly protein involved in the regulation of cellular Fe homeostasis. However, the biological function of FAM96A in vivo is still incompletely defined. Here, we tested the role of FAM96A in regulating organismal Fe metabolism, which is relevant to obesity and adipose tissue homeostasis. We found that in mice genetically lacking FAM96A globally, intracellular Fe homeostasis was interrupted in both white and brown adipocytes, but the systemic Fe level was normal. FAM96A deficiency led to adipocyte hypertrophy and organismal energy expenditure reduction even under nonobesogenic normal chow diet-fed conditions. Mechanistically, FAM96A deficiency promoted mechanistic target of rapamycin (mTOR) signaling in adipocytes, leading to an elevation of de novo lipogenesis and, therefore, fat mass accumulation. Furthermore, it also caused mitochondrial defects, including defects in mitochondrial number, ultrastructure, redox activity, and metabolic function in brown adipocytes, which are known to be critical for the control of energy balance. Moreover, adipocyte-selective FAM96A knockout partially phenocopied global FAM96A deficiency with adipocyte hypertrophy and organismal energy expenditure defects but the mice were resistant to high-fat diet-induced weight gain. Thus, FAM96A in adipocytes may autonomously act as a critical gatekeeper of organismal energy balance by coupling Fe metabolism to adipose tissue homeostasis.

A tyrosine catabolic intermediate 4-hydroxyphenylpyruate attenuates murine endotoxic shock by blocking NLRP3 inflammasome activation.

The metabolic alterations of amino acid metabolism are closely associated with inflammatory response. However, relatively little is known about the roles of phenylalanine (Phe)/tyrosine (Tyr) catabolites during inflammation. Nitisinone (NTBC) is an orphan drug used to treat hereditary tyrosinemia type I potentially by changing Phe/Tyr metabolic flow. In this study, we used NTBC as a tool to investigate the potential role of the Phe/Tyr catabolic pathway in inflammatory responses. We found that NTBC was effective in tempering the bacterial endotoxin lipopolysaccharide (LPS)-induced septic shock in mice. Mechanistically, the protective effect was related to the accumulation of a Phe/Tyr catabolic intermediate, 4-hydroxyphenylpyruvate (4-HPP), induced by the NTBC treatment. 4-HPP could inhibit NLRP3 inflammasome priming and activation processes and therefore reduce IL-1ß release and pyroptosis. Like NTBC, 4-HPP was also effective in attenuating endotoxic shock in mice. Our results suggest the Phe/Tyr catabolic pathway as a potential immunoregulatory hub that may be exploited therapeutically to alleviate inflammation.

The HVEM-BTLA Immune Checkpoint Restrains Murine Chronic Cholestatic Liver Injury by Regulating the Gut Microbiota.

The herpes virus entry mediator (HVEM) is an immune checkpoint molecule regulating immune response, but its role in tissue repair remains unclear. Here, we reported that HVEM deficiency aggravated hepatobiliary damage and compromised liver repair after 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced injury. A similar phenotype was observed in B and T lymphocyte attenuator (BTLA)-deficient mice. These were correlated with impairment of neutrophil accumulation in the liver after injury. The hepatic neutrophil accumulation was regulated by microbial-derived secondary bile acids. HVEM-deficient mice had reduced ability to deconjugate bile acids during DDC-feeding, suggesting a gut microbiota defect. Consistently, both HVEM and BTLA deficiency had dysregulated intestinal IgA responses targeting the gut microbes. These results suggest that the HVEM-BTLA signaling may restrain liver injury by regulating the gut microbiota.

A Murine Commensal Protozoan Influences Host Glucose Homeostasis by Facilitating Free Choline Generation.

Recent progress indicates that the gut microbiota plays important role in regulating the host's glucose homeostasis. However, the mechanisms remain unclear. Here, we reported that one integral member of the murine gut microbiota, the protozoan Tritrichomonas musculis could drive the host's glucose metabolic imbalance. Using metabolomics analysis and in vivo assays, we found that mechanistically this protozoan influences the host glucose metabolism by facilitating the production of a significant amount of free choline. Free choline could be converted sequentially by choline-utilizing bacteria and then the host to a final product trimethylamine N-oxide, which promoted hepatic gluconeogenesis. Together, our data reveal a previously underappreciated gut eukaryotic microorganism by working together with other members of microbiota to influence the host's metabolism. Our study underscores the importance and prevalence of metabolic interactions between the gut microbiota and the host in modulating the host's metabolic health. IMPORTANCE Blood glucose levels are important for human health and can be influenced by gut microbes. However, its mechanism of action was previously unknown. In this study, researchers identify a unique member of the gut microbes in mice that can influence glucose metabolism by promoting the host's ability to synthesis glucose by using nonglucose materials. This is because of its ability to generate the essential nutrient choline, and choline, aided by other gut bacteria and the host, is converted to trimethylamine N-oxide, which promotes glucose production. These studies show how gut microbes promote metabolic dysfunction and suggest novel approaches for treating patients with blood glucose abnormality.

LIGHT (TNFSF14) inhibits glucose uptake of adipocytes by downregulating GLUT4 expression via AKT signaling pathway.

Glucose homeostasis of adipocytes could be regulated by immune-adipose crosstalk. In order to investigate the effects of Lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells (LIGHT) on glucose metabolism, we performed the present study. Our results showed that LIGHT deficiency improved glucose tolerance and enhanced glucose consumption of inguinal white adipose tissue (iWAT) under high fat diet. Consistently, Light overexpression could inhibit glucose uptake during the process of white adipogenesis. Mechanistically, LIGHT interacted with lymphotoxin-ß receptor (LTßR) to attenuate AKT pathway leading to downregulation of glucose transporter-4 (GLUT4) expression, which resulted in glucose uptake inhibition. In summary, our findings revealed LIGHT-LTßR-AKT-GLUT4 axis as a regulator of glucose uptake in adipose tissue, which suggested the pivotal role of LIGHT in maintaining glucose homeostasis.

Aurka deficiency in the intestinal epithelium promotes age-induced obesity via propionate-mediated AKT activation.

Aurora-A kinase, a serine/threonine mitotic kinase involved in mitosis, is overexpressed in several human cancers. A recent study showed that Aurora-A mediates glucose metabolism via SOX8/FOXK1 in ovarian cancer. However, the roles of Aurora-A in metabolic diseases remain unclear. This study found that Aurka loss in the intestinal epithelium promoted age-induced obesity and enlargement of lipid droplets in parallel with an increase in infiltrated macrophages in the white adipocyte tissue (WAT) of male mice. Moreover, loss of Aurka induced the expression of lipid metabolism regulatory genes, including acetyl-coenzyme A carboxylase 1 (Acc1), in association with an increase in the levels of p-AKT in the intestinal epithelium as well as WAT. Blockade of AKT activation reduced the expression of lipid metabolism regulatory genes. In subsequent experiments, we found that the Firmicutes abundance and the levels of short-chain fatty acids (SCFAs) in the gut were dramatically increased in Aurkaf/+;VillinCre/+ mice compared with Aurkaf/+ mice. Additionally, propionate increased the phosphorylation of AKT in vitro. These observations indicated that Aurka loss in the intestinal epithelium contributed to gut microbiota dysbiosis and higher levels of SCFAs, especially propionate, leading to AKT activation and lipid metabolism regulatory gene expression, which in turn promoted age-induced obesity.

A Transcriptomic Analysis Reveals Novel Patterns of Gene Expression During 3T3-L1 Adipocyte Differentiation.

BACKGROUND: Obesity is characterized by increased adipose tissue mass that results from increased fat cell size (hypertrophy) and number (hyperplasia). The molecular mechanisms that govern the regulation and differentiation of adipocytes play a critical role for better understanding of the pathological mechanism of obesity. However, the mechanism of adipocyte differentiation is still unclear. OBJECTIVE: The present study aims to compare the gene expression changes during adipocyte differentiation in the transcriptomic level, which may help to better understand the mechanism of adipocyte differentiation. METHODS: RNA sequencing (RNA-seq) technology, GO and KEGG analysis, quantitative RT-PCR, and oil red O staining methods were used in this study. RESULTS: A lot of genes were up- or down-regulated between each two differentiation stages of 3T3-L1 cells. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that lipid metabolism and oxidation-reduction reaction were mainly involved in the whole process of adipocyte differentiation. Decreased immune response and cell cycle adhesion occurred in the late phase of adipocyte differentiation, which was demonstrated by divergent expression pattern analysis. Moreover, quantitative RT-PCR results showed that the mRNA expression levels of Trpv4, Trpm4, Trpm5, and Trpm7 were significantly decreased in the differentiated adipocytes. On the other hand, the mRNA expression levels of Trpv1, Trpv2, Trpv6, and Trpc1 were significantly increased in the differentiated adipocytes. Besides, the mRNA expressions of TRPV2 and TRPM7 were also significantly increased in subcutaneous white adipose tissue from diet-induced mice. In addition, the activation of TRPM7, TRPV1, and TRPV2 suppressed the differentiation of adipocytes. CONCLUSION: These data present the description of transcription profile changes during adipocyte differentiation and provides an in-depth analysis of the possible mechanisms of adipocyte differentiation. These data offer new insight into the understanding of the mechanisms of adipocyte differentiation.

The intestinal microbial metabolite desaminotyrosine is an anti-inflammatory molecule that modulates local and systemic immune homeostasis.

It is considered that intestinal barrier dysfunction and systemic endotoxemia drive obesity and its related complications. However, what causes barrier dysfunction remains to be elucidated. Here, we showed that the gut microbiota from high-fat diet (HFD)-fed mice had impaired ability to degrade dietary flavonoids, and in correspondence, the microbial-derived flavonoid metabolite desaminotyrosine (DAT) was reduced. Supplementation of DAT in the drinking water was able to counter the HFD-induced body fat mass accumulation and body weight increment. This is correlated with the role of DAT in maintaining mucosal immune homeostasis to protect barrier integrity. DAT could attenuate dextran sodium sulfate (DSS)-induced mucosal inflammation in a type I interferon signal-dependent manner. Furthermore, intraperitoneal injection of DAT-protected mice from bacterial endotoxin-induced septic shock. Together, we identified DAT as a gut microbiota-derived anti-inflammatory metabolite that functions to modulate local and systemic immune homeostasis. Our data support the notion of dysbiosis being an important driving force of mucosal barrier dysfunction and systemic metabolic complications.

Commensal Bacteria Impact a Protozoan's Integration into the Murine Gut Microbiota in a Dietary Nutrient-Dependent Manner.

Our current understanding of the host-microbiota interaction in the gut is dominated by studies focused primarily on prokaryotic bacterial communities. However, there is an underappreciated symbiotic eukaryotic protistic community that is an integral part of mammalian microbiota. How commensal protozoan bacteria might interact to form a stable microbial community remains poorly understood. Here, we describe a murine protistic commensal, phylogenetically assigned as Tritrichomonas musculis, whose colonization in the gut resulted in a reduction of gut bacterial abundance and diversity in wild-type C57BL/6 mice. Meanwhile, dietary nutrient and commensal bacteria also influenced the protozoan's intestinal colonization and stability. While mice fed a normal chow diet had abundant T. musculis organisms, switching to a Western-type high-fat diet led to the diminishment of the protozoan from the gut. Supplementation of inulin as a dietary fiber to the high-fat diet partially restored the protozoan's colonization. In addition, a cocktail of broad-spectrum antibiotics rendered permissive engraftment of T. musculis even under a high-fat, low-fiber diet. Furthermore, oral administration of Bifidobacterium spp. together with dietary supplementation of inulin in the high-fat diet impacted the protozoan's intestinal engraftment in a bifidobacterial species-dependent manner. Overall, our study described an example of dietary-nutrient-dependent murine commensal protozoan-bacterium cross talk as an important modulator of the host intestinal microbiome.IMPORTANCE Like commensal bacteria, commensal protozoa are an integral part of the vertebrate intestinal microbiome. How protozoa integrate into a commensal bacterium-enriched ecosystem remains poorly studied. Here, using the murine commensal Tritrichomonas musculis as a proof of concept, we studied potential factors involved in shaping the intestinal protozoal-bacterial community. Understanding the rules by which microbes form a multispecies community is crucial to prevent or correct microbial community dysfunctions in order to promote the host's health or to treat diseases.

HBV upregulates AP-1 complex subunit mu-1 expression via the JNK pathway to promote proliferation of liver cancer cells.

Although hepatitis B virus (HBV) infection is responsible for liver cancer, the exact mechanism of its action remains unclear. µ1 adaptin is an intrinsic part of the clathrin adaptor AP-1 complex. In addition to its canonical biological function that involves cargo sorting and vesicular transport, recent studies have demonstrated that µ1 adaptin participates in cell growth and proliferation. The aim of the present study was to investigate the effects of the clathrin adaptor AP-1 complex subunit mu-1 (AP1M1) on liver cancer cell proliferation. The present study reports for the first time that AP1M1 is upregulated in the HBV-transfected HepG2.215 liver cancer cells. Silencing of AP1M1 in HepG2.215 cells suppressed their proliferation, while the overexpression of AP1M1 in HepG2 cells promoted cell proliferation. The data suggested that AP1M1 is one of the crucial factors involved in the progression of liver cancer caused by HBV infection. In addition, it was demonstrated that HBV facilitated AP1M1 expression in a JNK-dependent manner. The increased expression levels of AP1M1 enhanced phosphorylation of protein kinase B and accelerated cell proliferation. Unraveling the effects of AP1M1 on liver cancer cell proliferation and the mechanism of AP1M1 transcriptional regulation may provide new therapeutic targets for HBV-positive liver cancer.

LIGHT/TNFSF14 signaling attenuates beige fat biogenesis.

The physiologic signals that regulate beige adipogenesis remain incompletely understood, especially those that limit browning and prevent overexpenditure of energy. In this study, the TNF family member cytokine lymphotoxin-like inducible protein that competes with glycoprotein D for herpesvirus entry on T cells (LIGHT), also known as TNF super family protein 14 (TNFSF14), can inhibit adipose precursor differentiation into beige adipocytes. In acute cold stress, LIGHT deficiency in mice accelerated browning in the subcutaneous white adipose tissue (scWAT). Further experiments showed that LIGHT interacting with lymphotoxin-ß receptor (LTßR) on adipose precursors blocked beige fat biogenesis. LTßR signals attenuated the JNK pathway, which contributed to their antibeiging effect. Blocking JNK activation using a small molecular inhibitor prevented cold-induced scWAT beiging. Furthermore, LIGHT/LTßR signals acted as an attenuator of white adipogenesis. LIGHT deficiency in mice promoted obesity during high-fat diet feeding. These findings identify the LIGHT axis as a regulator of adipose tissue homeostasis and suggest that LIGHT signaling functions as a mechanism to divert energy in favor of immune activation.-Kou, Y., Liu, Q., Liu, W., Sun, H., Liang, M., Kong, F., Zhang, B., Wei, Y., Liu, Z., Wang, Y. LIGHT/TNFSF14 signaling attenuates beige fat biogenesis.

MiR-409-3p and MiR-1896 co-operatively participate in IL-17-induced inflammatory cytokine production in astrocytes and pathogenesis of EAE mice via targeting SOCS3/STAT3 signaling.

Th17 cells and interleukin-17 (IL-17) have been found to play an important role in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). Response to IL-17, reactive astrocytes accompany with immune cells infiltration and axonal damage in MS/EAE. However, the role and the regulatory mechanism of IL-17-activated astrocytes in inflammation and in the EAE process still remain largely unknown. Here, we elucidated that miR-409-3p and miR-1896, as co-upregulated microRNAs in activated astrocytes and in EAE mice, targeted suppressor of cytokine signaling proteins 3 (SOCS3). Overexpression of miR-409-3p or miR-1896 significantly reduced SOCS3 expression and increased phosphorylation of STAT3 as well as induced the inflammatory cytokines production (IL-1ß, IL-6, IP-10, MCP-1, and KC), CD4+ T cells migration and demyelination, in turn aggravating EAE development. Importantly, the effects of co-overexpression of miR-409-3p and miR-1896 in vitro or in vivo are strongly co-operative. In contrast, simultaneously silenced miR-409-3p and miR-1896 co-operatively ameliorates inflammation and demyelination in the central nervous system of EAE mice. Collectively, our findings highlight that miR-409-3p and miR-1896 co-ordinately promote the production of inflammatory cytokines in reactive astrocytes through the SOCS3/STAT3 pathway and enhance reactive astrocyte-directed chemotaxis of CD4+ T cells, leading to aggravate pathogenesis in EAE mice. Co-inhibition of miR-409-3p and miR-1896 may be a therapeutic target for treating MS and neuroinflammation.

The Probiotic Effectiveness in Preventing Experimental Colitis Is Correlated With Host Gut Microbiota.

Current evidence to support extensive use of probiotics in inflammatory bowel disease is limited and factors that contribute to the inconsistent effectiveness of clinical probiotic therapy are not completely known. Here, we used Bifidobacterium longum JDM 301 as a model probiotic to study potential factors that may influence the effect of probiotics in experimental colitis. We found that the effect of B. longum JDM 301 in tempering experimental colitis varied across individual mice even with the same genetic background. The probiotic efficacy was highly correlated with the host gut microbial community features. Consumption of a diet rich in fat could exacerbate mucosal injury-induced colitis but could not change the host responsiveness to B. longum JDM 301 treatment, suggesting of potential mechanistic differences between regulating colitis pathogenesis, and modulating probiotic efficacies by the gut microbiota. Together, our results suggest that personalized microbiome features may modify the probiotic therapeutic effect and support the idea of personalized probiotic medicine in inflammatory bowel disease.

Protective Effects of Bifidobacterial Strains Against Toxigenic Clostridium difficile.

Probiotics might offer an attractive alternative to prevent and control Clostridium difficile (C. difficile) infection (CDI). Limited information is available on the ability of commercially used bifidobacterial strains to inhibit C. difficile. This study examined the anti-clostridial effects of Bifidobacterium longum JDM301, a widely used commercial probiotic strain in China, in vitro and in vivo. In vitro evaluation revealed a significant reduction in C. difficile counts when JDM301 was co-cultured with C. difficile, which was correlated with the significant decrease in clostridial toxin titres (TcdA and TcdB). Furthermore, the cell-free culture supernatants (CFS) of JDM301 inhibited C. difficile growth and degraded TcdA and TcdB. Notably, the results showed that acid pH promoted the degradation of TcdA by CFS from JDM301. Furthermore, comparative studies among 10 B. longum strains were performed, which showed that the inhibitory effect of CFS from JDM301 was similar with the other 8 B. longum strains and higher than strain BLY1. However, when it was neutralized, the significant different was lost. When present together, it was suggested that the acid pH induced by probiotics not only played important roles in the growth inhibition against C. difficile resulting in the reduction of toxins titres, but also directly promoted the degradation of clostridial toxin. In vivo studies proved that JDM301 partially relieved damage to tissues caused by C. difficile and also decreased the number of C. difficile and toxin levels. In summary, our results demonstrated that the commercial strain, JDM301 could be considered a probiotic able to exert anti-toxin capability and most of the CFS from Bifidobacterium were able to inhibit the growth of C. difficile, depending on acid pH. These results highlighted a potential that JDM301 could be helpful in preventing CDI and that most of the bifidobacterial strains could (at least partially) exert protective effects by reducing toxin titres through growth inhibition against toxigenic C. difficile.

HBX protein promotes LASP-1 expression through activation of c-Jun in human hepatoma cells.

LIM and SH3 domain protein 1 (LASP-1) is known to participate in the progression of hepatocellular carcinoma (HCC). We previously showed that ectopic expression of hepatitis B virus (HBV) X protein (HBX) enhanced the expression of LASP-1, which promoted proliferation and migration of HCC cells. Here, we further demonstrated the molecular mechanism underlying upregulation of LASP-1, mediated by HBX, in HBV-infected HCC cells. Through a luciferase activity assay, we discovered that the LASP-1 promoter region regulated by HBX contained an AP-1 binding element in human hepatoma cells. Interestingly, c-Jun, one subunit of AP-1, was mainly responsible for activation, mediated by HBX, of the LASP-1 promoter. Furthermore, HBX was shown not only to interact with phosphorylated c-Jun in HCC cells but also to activate c-Jun by increasing the activation of PI3-K/JNK signaling. Chromatin immunoprecipitation (ChIP) assay demonstrated that HBX was capable of binding to the LASP-1 promoter with c-Jun. Further, the expression levels of HBX were shown to be significantly positively correlated with that of LASP-1 and phosphorylatedc-Jun in HBV-related HCC tissues by immunohistochemistry analysis. In addition, the N-terminus of HBX was found to be responsible for the activation of c-Jun, as well as the expression of LASP-1. Taken together, these results suggest that HBX contributes to LASP-1 expression via the activation of c-Jun to increase the promoter activity of LASP-1 in HBV-related HCC cells.

Analysis of Long Noncoding RNA and mRNA Expression Profiles in IL-9-Activated Astrocytes and EAE Mice.

BACKGROUND/AIMS: Multiple sclerosis (MS) is an autoimmune disease in the central nervous system associated with demyelination and axonal injury. Astrocyte activation is involved in the pathogenesis of MS and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This study was designed to find potential lncRNAs in EAE mice and activated astrocytes. METHODS: we performed microarray analysis of lncRNAs from the brain tissues of EAE mice and primary mouse astrocytes treated with IL-9(50 ng/ml). 12 lncRNAs were validated through real-time PCR. Gene ontology and KEGG pathway analysis were applied to explore the potential functions of lncRNAs. RESULTS: Differentially expressed 3300 lncRNAs and 3250 mRNAs were in the brain tissues of EAE mice, and 3748 lncRNAs and 3332 mRNAs were in activated astrocytes. Notably, there were 2 co-up-regulated lncRNAs and 3 co-down-regulated lncRNAs both in the brain tissues of EAE mice and in activated astrocytes, including Gm14005, Gm12478, mouselincRNA1117, AK080435, and mouselincRNA0681, which regulate the ER calcium flux kinetics, zinc finger protein and cell apoptosis. Similarly, there were 7 mRNAs co-up-regulated and 2 mRNAs co-down-regulated both in vivo and in vitro. Gene ontology and KEGG pathway analysis showed that the biological functions of differentially expressed mRNAs were associated with metabolism, development and inflammation. The results of realtime PCR validation were consistent with the data from the microarrays. CONCLUSIONS: Our data uncovered the expression profiles of lncRNAs and mRNAs in vivo and in vitro, which may help delineate the mechanisms of astrocyte activation during MS/EAE process.

Bioinformatics analysis of the proteins interacting with LASP-1 and their association with HBV-related hepatocellular carcinoma.

LIM and SH3 domain protein (LASP-1) is responsible for the development of several types of human cancers via the interaction with other proteins; however, the precise biological functions of proteins interacting with LASP-1 are not fully clarified. Although the role of LASP-1 in hepatocarcinogenesis has been reported, the implication of LASP-1 interactors in HBV-related hepatocellular carcinoma (HCC) is not clearly evaluated. We obtained information regarding LASP-1 interactors from public databases and published studies. Via bioinformatics analysis, we found that LASP-1 interactors were related to distinct molecular functions and associated with various biological processes. Through an integrated network analysis of the interaction and pathways of LASP-1 interactors, cross-talk between different proteins and associated pathways was found. In addition, LASP-1 and several its interactors are significantly altered in HBV-related HCC through microarray analysis and could form a complex co-expression network. In the disease, LASP-1 and its interactors were further predicted to be regulated by a complex interaction network composed of different transcription factors. Besides, numerous LASP-1 interactors were associated with various clinical factors and related to the survival and recurrence of HBV-related HCC. Taken together, these results could help enrich our understanding of LASP-1 interactors and their relationships with HBV-related HCC.