Showing papers in "Journal of Molecular Cell Biology in 2016"

Adiponectin, the past two decades

Abstract: Adiponectin is an adipocyte-specific factor, first described in 1995. Over the past two decades, numerous studies have elucidated the physiological functions of adiponectin in obesity, diabetes, inflammation, atherosclerosis, and cardiovascular disease. Adiponectin, elicited through cognate receptors, suppresses glucose production in the liver and enhances fatty acid oxidation in skeletal muscle, which together contribute to a beneficial metabolic action in whole body energy homeostasis. Beyond its role in metabolism, adiponectin also protects cells from apoptosis and reduces inflammation in various cell types via receptor-dependent mechanisms. Adiponectin, as a fat-derived hormone, therefore fulfills a critical role as an important messenger to communicate between adipose tissue and other organs. A better understanding of adiponectin actions, including the pros and cons, will advance our insights into basic mechanisms of metabolism and inflammation, and potentially pave the way toward novel means of pharmacological intervention to address pathophysiological changes associated with diabetes, atherosclerosis, and cardiometabolic disease.

Adiponectin signaling and function in insulin target tissues

Abstract: Obesity-linked type 2 diabetes is one of the paramount causes of morbidity and mortality worldwide, posing a major threat on human health, productivity, and quality of life. Despite great progress made towards a better understanding of the molecular basis of diabetes, the available clinical counter-measures against insulin resistance, a defect that is central to obesity-linked type 2 diabetes, remain inadequate. Adiponectin, an abundant adipocyte-secreted factor with a wide-range of biological activities, improves insulin sensitivity in major insulin target tissues, modulates inflammatory responses, and plays a crucial role in the regulation of energy metabolism. However, adiponectin as a promising therapeutic approach has not been thoroughly explored in the context of pharmacological intervention, and extensive efforts are being devoted to gain mechanistic understanding of adiponectin signaling and its regulation, and reveal therapeutic targets. Here, we discuss tissue- and cell-specific functions of adiponectin, with an emphasis on the regulation of adiponectin signaling pathways, and the potential crosstalk between the adiponectin and other signaling pathways involved in metabolic regulation. Understanding better just why and how adiponectin and its downstream effector molecules work will be essential, together with empirical trials, to guide us to therapies that target the root cause(s) of type 2 diabetes and insulin resistance.

Adiponectin: a versatile player of innate immunity

Abstract: Adiponectin acts as a key regulator of the innate immune system and plays a major role in the progression of inflammation and metabolic disorders. Macrophages and monocytes are representative components of the innate immune system, and their proliferation, plasticity, and polarization are a key component of metabolic adaption. Innate-like lymphocytes such as group 2 innate lymphoid cells (ILC2s), natural killer T (NKT) cells, and gamma delta T (γδ T) cells are also members of the innate immune system and play important roles in the development of obesity and its related diseases. Adiponectin senses metabolic stress and modulates metabolic adaption by targeting the innate immune system under physiological and pathological conditions. Defining the mechanisms underlying the role of adiponectin in regulating innate immunity is crucial to adiponectin-based therapeutic intervention.

Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

Abstract: Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance.

Adipocyte-derived microvesicles from obese mice induce M1 macrophage phenotype through secreted miR-155

Abstract: The pro-inflammatory profile of M1 macrophage accumulation in adipose tissue is a central event leading to the metabolic complications of obesity. However, the mechanisms by which M1 macrophages are enriched in adipose tissue during weight gain remain incompletely understood. Here, we investigated the effects of adipocyte-derived microvesicles (ADM) on modulating macrophage phenotype in mice and explored the involved molecular signalling pathways. We found that, compared with ADM from lean mice (SD ADM), ADM from obese mice (HFD ADM) significantly enhanced M1 marker expression. The quantitative RT-PCR assay demonstrated that miR-155 was upregulated in both HFD ADM and HFD ADM-treated macrophages. By depleting miR-155 expression in HFD ADM and increasing miR-155 level in SD ADM, we further illustrated that miR-155 in ADM-induced M1 macrophage polarization. Functionally, in contrast to SD ADM, HFD ADM significantly decreased the protein level of SOCS1, a proven miR-155 target, leading to activation of STAT1, and suppression of STAT6 signalling; these effects were reversed by silencing miR-155 in HFD ADM. Furthermore, the supernatant of bone marrow-derived macrophages pre-stimulated with miR-155-bearing ADM interfered with insulin signalling and insulin-induced glucose uptake in adipocytes. Collectively, these results provide the first evidence that M1 macrophage polarization can be mediated by miR-155-bearing ADM, which reciprocally regulates insulin signalling and glucose uptake in adipocytes. Our study reveals a novel mechanism through which obesity induces an imbalance in the M1-to-M2 macrophage ratio in adipose tissue, thus causing chronic inflammation and local insulin resistance.

Interleukin-38 is released from apoptotic cells to limit inflammatory macrophage responses

Abstract: Different modes of cell death regulate immunity. Whereas necrotic (necroptotic, pyroptotic) cell death triggers inflammation, apoptosis contributes to its resolution. Interleukin-1 (IL-1) family cytokines are key players in this interaction. A number of IL-1 family cytokines are produced by necrotic cells to induce sterile inflammation. However, release of IL-1 family proteins from apoptotic cells to regulate inflammation was not described. Here we show that IL-38, a poorly characterized IL-1 family cytokine, is produced selectively by human apoptotic cells to limit inflammation. Depletion of IL-38 in apoptotic cells provoked enhanced IL-6 and IL-8 levels and AP1 activation in co-cultured human primary macrophages, subsequently inducing Th17 cell expansion at the expense of IL-10-producing T cells. IL-38 was N-terminally processed in apoptotic cells to generate a mature cytokine with distinct properties. Both full-length and truncated IL-38 bound to X-linked interleukin-1 receptor accessory protein-like 1 (IL1RAPL1). However, whereas the IL-38 precursor induced an increase in IL-6 production by human macrophages, truncated IL-38 reduced IL-6 production by attenuating the JNK/AP1 pathway downstream of IL1RAPL1. In conclusion, we identified a mechanism of apoptotic cell-dependent immune regulation requiring IL-38 processing and secretion, which might be relevant in resolution of inflammation, autoimmunity, and cancer.

SNORD126 promotes HCC and CRC cell growth by activating the PI3K-AKT pathway through FGFR2.

Abstract: Small nucleolar RNA (snoRNA) dysfunctions have been associated with cancer development. SNORD126 is an orphan C/D box snoRNA that is encoded within introns 5-6 of its host gene, cyclin B1-interacting protein 1 (CCNB1IP1). The cancer-associated molecular mechanisms triggered by SNORD126 are not fully understood. Here, we demonstrate that SNORD126 is highly expressed in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) patient samples. SNORD126 increased Huh-7 and SW480 cell growth and tumorigenicity in nude mice. Knockdown of SNORD126 inhibited HepG2 and LS174T cell growth. We verified that SNORD126 was not processed into small RNAs with miRNA activity. Moreover, SNORD126 did not show a significant expression correlation with CCNB1IP1 in HCC samples or regulate CCNB1IP1 expression. Our gene expression profile analysis indicated that SNORD126-upregulated genes frequently mapped to the PI3K-AKT pathway. SNORD126 overexpression increased the levels of phosphorylated AKT, GSK-3β, and p70S6K and elevated fibroblast growth factor receptor 2 (FGFR2) expression. siRNA-mediated knockdown or AZD4547-mediated inactivation of FGFR2 in SNORD126-overexpressing Huh-7 cells inhibited AKT phosphorylation and suppressed cell growth. These findings indicate an oncogenic role for SNORD126 in cancer and suggest its potential as a therapeutic target.

Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells

Abstract: Although the conserved AAA ATPase and bromodomain factor, ATAD2, has been described as a transcriptional co-activator upregulated in many cancers, its function remains poorly understood. Here, using a combination of ChIP-seq, ChIP-proteomics, and RNA-seq experiments in embryonic stem cells where Atad2 is normally highly expressed, we found that Atad2 is an abundant nucleosome-bound protein present on active genes, associated with chromatin remodelling, DNA replication, and DNA repair factors. A structural analysis of its bromodomain and subsequent investigations demonstrate that histone acetylation guides ATAD2 to chromatin, resulting in an overall increase of chromatin accessibility and histone dynamics, which is required for the proper activity of the highly expressed gene fraction of the genome. While in exponentially growing cells Atad2 appears dispensable for cell growth, in differentiating ES cells Atad2 becomes critical in sustaining specific gene expression programmes, controlling proliferation and differentiation. Altogether, this work defines Atad2 as a facilitator of general chromatin-templated activities such as transcription.

Mismatch repair proteins recruit DNA methyltransferase 1 to sites of oxidative DNA damage

Abstract: At sites of chronic inflammation, epithelial cells are exposed to high levels of reactive oxygen species and undergo cancer-associated DNA methylation changes, suggesting that inflammation may initiate epigenetic alterations. Previously, we demonstrated that oxidative damage causes epigenetic silencing proteins to become part of a large complex that is localized to GC-rich regions of the genome, including promoter CpG islands that are epigenetically silenced in cancer. However, whether these proteins were recruited directly to damaged DNA or during the DNA repair process was unknown. Here we demonstrate that the mismatch repair protein heterodimer MSH2-MSH6 participates in the oxidative damage-induced recruitment of DNA methyltransferase 1 (DNMT1) to chromatin. Hydrogen peroxide treatment induces the interaction of MSH2-MSH6 with DNMT1, suggesting that the recruitment is through a protein-protein interaction. Importantly, the reduction in transcription for genes with CpG island-containing promoters caused by oxidative damage is abrogated by knockdown of MSH6 and/or DNMT1. Our findings provide evidence that the role of DNMT1 at sites of oxidative damage is to reduce transcription, potentially preventing transcription from interfering with the repair process. This study uniquely brings together several factors that are known to contribute to colon cancer, namely inflammation, mismatch repair proteins, and epigenetic changes.

The FGF21–adiponectin axis in controlling energy and vascular homeostasis

Abstract: Whole-body energy metabolism and cardiovascular homeostasis are tightly controlled processes that involve highly coordinated crosstalk among distal organs. This is mainly achieved by a large number of hormones released from each organ. Among them, fibroblast growth factor 21 (FGF21) and adiponectin have recently gained considerable attention, since both of them possess multiple profound protective effects against a myriad of cardio-metabolic disorders. Despite their distinct structures and production sites, these two hormones share striking functional similarity. This dichotomy is recently reconciled by the demonstration of the FGF21-adiponectin axis. In adipocytes, both transcription and secretion of adiponectin are strongly induced by FGF21, which is partially dependent on PPARγ activity. Furthermore, the glucose-lowering, lipid-clearing, and anti-atherosclerotic functions of FGF21 are diminished in adiponectin-null mice, suggesting that adiponectin serves as an obligatory mediator of FGF21-elicited metabolic and vascular benefits. However, in both animals and human subjects with obesity, circulating FGF21 levels are increased whereas plasma adiponectin concentrations are reduced, perhaps due to FGF21 resistance, suggesting that dysfunctional FGF21-adiponectin axis is an important contributor to the pathogenesis of obesity-related cardio-metabolic syndrome. The FGF21-adiponectin axis protects against a cluster of cardio-metabolic disorders via mediating multi-organ communications, and is a promising target for therapeutic interventions of these chronic diseases.

Post-translational O-GlcNAcylation is essential for nuclear pore integrity and maintenance of the pore selectivity filter

Abstract: O-glycosylation of the nuclear pore complex (NPC) by O-linked N-acetylglucosamine (O-GlcNAc) is conserved within metazoans. Many nucleoporins (Nups) comprising the NPC are constitutively O-GlcNAcylated, but the functional role of this modification remains enigmatic. We show that loss of O-GlcNAc, induced by either inhibition of O-GlcNAc transferase (OGT) or deletion of the gene encoding OGT, leads to decreased cellular levels of a number of natively O-GlcNAcylated Nups. Loss of O-GlcNAc enables increased ubiquitination of these Nups and their increased proteasomal degradation. The decreased half-life of these deglycosylated Nups manifests in their gradual loss from the NPC and a downstream malfunction of the nuclear pore selective permeability barrier in both dividing and post-mitotic cells. These findings define a critical role of O-GlcNAc modification of the NPC in maintaining its composition and the function of the selectivity filter. The results implicate NPC glycosylation as a regulator of NPC function and reveal the role of conserved glycosylation of the NPC among metazoans.

Autoubiquitination of TRIM26 links TBK1 to NEMO in RLR-mediated innate antiviral immune response

Abstract: The transcription factors IRF3 and NF-κB are required for the expression of many genes involved in antiviral innate immune response, including type I interferons (IFNs) and proinflammatory cytokines. It is well established that TBK1 is an essential kinase engaged downstream of multiple pattern-recognition receptors (PRRs) to mediate IRF3 phosphorylation and activation, whereas the precise mechanisms of TBK1 activation have not been fully elucidated yet. Here, we identified tripartite motif 26 (TRIM26) as an important regulator for RNA virus-triggered innate immune response. Knockdown of TRIM26 impaired virus-triggered IRF3, NF-κB activation, IFN-β induction, and cellular antiviral response. TRIM26 was physically associated with TBK1 independent of viral infection. As an E3 ligase, TRIM26 underwent autoubiquitination upon viral infection. Ubiquitinated TRIM26 subsequently associated with NEMO, thus bridging TBK1-NEMO interaction, which is critical for the recruitment of TBK1 to the VISA signalsome and activation of TBK1. Our findings suggest that TRIM26 is an important regulator of innate immune responses against RNA viruses, which functions by bridging TBK1 to NEMO and mediating the activation of TBK1.

BTG4 is a key regulator for maternal mRNA clearance during mouse early embryogenesis

Abstract: Dear Editor, The maternal to zygotic transition (MZT) is a crucial process in the early development of almost all animals, during which maternal mRNAs are degraded and the zygotic genome is activated (Li et al., 2013). How maternal mRNAs are degraded is one of the long-standing questions in the field of developmental and reproductive biology. Recently, high-throughput sequencing and genetic studies have determined that the elimination of maternal mRNAs is accomplished by two modes: the first mode is dependent on maternally encoded transcripts, while the second mode relies on zygotic transcription (Yartseva and Giraldez, 2015). The first mode is well characterized in Drosophila, in which the maternally encoded RNA-binding proteins SMAUG and PUMILIO are important mediators of maternal mRNA clearance (Semotok et al., 2005; Gerber et al., 2006). The second mode is exemplified by zygotically transcribed miR-430 in zebrafish that directly targets and triggers the deadenylation and subsequent clearance of maternal mRNAs (Giraldez et al., 2006). Despite these encouraging findings in model animals of lower species, the mechanisms governing the selective maternal mRNA clearance are still largely unclear in mammals. Deadenylation is the first and often ratelimiting step accounting for mRNA turnover (Garneau et al., 2007). The CCR4–NOT complex plays predominant roles in mRNA deadenylation, whereby it is involved in the SMAUGand PUMILIO-mediated maternal mRNA clearance in Drosophila (Semotok et al., 2005; Gerber et al., 2006). Previous studies have shown that the anti-proliferative Tob/BTG protein family members are important players in CCR4– NOT-mediated mRNA deadenylation and subsequent degradation (Winkler, 2010). To investigate the potential roles of Tob/ BTG proteins during mouse MZT, we first systematically analyzed the expression patterns of all Tob/BTG members (Tob1, Tob2, and Btg1–4) during this process by using RNA-seq data sets that are publicly available. Strikingly, among all the six members, Btg4, the main functional domain of which is highly conserved among vertebrates, showed an absolutely dominant and specific expression pattern during the MZT (Supplementary Figures S1 and S2A). Next, we examined the spatio-temporal expression pattern of Btg4 by quantitative real-time PCR (qRT-PCR) and western blot. Btg4 was exclusively present in ovaries and testes (Supplementary Figure S2B). In oocytes and preimplantation embryos, Btg4 mRNA was highly expressed in germinal vesicle stage (GV) oocytes and gradually perished during mouse preimplantation development, decreasing by ~90% by the 2cell stage (Figure 1A). At the protein level, however, the translation of Btg4 was largely initiated in metaphase II (MII) oocytes and was peaked at the 1-cell (1C) stage (Figure 1B, Supplementary Figure S2C), depending on the cytoplasmic polyadenylation elements and polyadenylation hexanucleotide AAUAAA sequence in the 3′ UTR (Supplementary Figure S3). To investigate the physiological role of Btg4, we depleted Btg4 using the CRISPR/Cas9 system that targets the first exon, and a targeted frame-shift mutant with a 116-bp deletion (Btg4) was successfully obtained (Supplementary Figure S4A and B). qRT-PCR and western blot showed that Btg4 was efficiently disrupted at both RNA and protein levels (Supplementary Figure S4C and D). Disruption of Btg4 had no effect on mouse viability and the fertility of male mice, but led to the infertility of female mice (Figure 1C). Histological analysis of paraffin sections from wild-type (WT) and Btg4 ovaries and superovulation experiments demonstrated that the oogenesis and fertilization of Btg4 oocytes were grossly normal (Supplementary Figure S5), but the development of embryos from Btg4 female mice was arrested at 1–2-cell stage (Figure 1D), indicating essential roles of Btg4 during the MZT of mouse early embryogenesis. We further performed transcriptome analysis on WT and Btg4 GV oocytes, MII oocytes, and 1C embryos (Supplementary Tables S1–S3). Although the transcriptome profile of Btg4 GV oocytes was comparable to that of WT, >46% (6401/13770) and 20% (2898/ 14058) mRNAs were upregulated in Btg4 MII oocytes and the resulting 1C embryos, respectively (Figure 1E). These findings were consistent with the temporal protein expression pattern and thus the functions of Btg4 during mouse preimplantation development. Moreover, all the 12 genes, generated by overlapping the top 50 significantly upregulated genes in MII oocytes and 1C embryos, could be well validated by qRT-PCR, except for one gene with a very low expression level (Figure 1F, Supplementary Figure S6). To test whether the aberrant upregulation of maternal transcripts is caused by the inefficient deadenylation of maternal mRNAs, poly(A) tail length (PAT) assay (Supplementary Figure S7A) was performed with several representative transcripts that were abundant in GV oocytes but significantly reduced in MII oocytes (Ma et al., 2015). The results showed that these transcripts in Btg4 MII oocytes and the resulting 1C embryos failed to be deadenylated and were thus resistant to degradation (Figure 1G and Supplementary Figure S7B). As the CCR4–NOT complex

E-selectin ligand complexes adopt an extended high-affinity conformation

Abstract: E-selectin is a cell-adhesion molecule of the vascular endothelium that promotes essential leukocyte rolling in the early inflammatory response by binding to glycoproteins containing the tetrasaccharide sialyl Lewis(x) (sLe(x)). Efficient leukocyte recruitment under vascular flow conditions depends on an increased lifetime of E-selectin/ligand complexes under tensile force in a so-called catch-bond binding mode. Co-crystal structures of a representative fragment of the extracellular E-selectin region with sLe(x) and a glycomimetic antagonist thereof reveal an extended E-selectin conformation, which is identified as a high-affinity binding state of E-selectin by molecular dynamics simulations. Small-angle X-ray scattering experiments demonstrate a direct link between ligand binding and E-selectin conformational transition under static conditions in solution. This permits tracing a series of concerted structural changes connecting ligand binding to conformational stretching as the structural basis of E-selectin catch-bond-mediated leukocyte recruitment. The detailed molecular view of the binding site paves the way for the design of a new generation of selectin antagonists. This is of special interest, since their therapeutic potential was recently demonstrated with the pan-selectin antagonists GMI-1070 (Rivipansel).

microRNA-129-5p, a c-Myc negative target, affects hepatocellular carcinoma progression by blocking the Warburg effect

Abstract: Deregulation of microRNAs (miRNAs) and c-Myc (Myc) contributes to hepatocellular carcinoma (HCC) progression, but how miRNAs and Myc regulate each other in hepatocarcinogenesis is still poorly understood. Using a functional screen, we identified miR-129-5p as a miRNA that inhibits HCC cell growth. miR-129-5p targets the mitochondrial matrix protein pyruvate dehydrogenase kinase 4 (PDK4), which leads to decreased phosphorylation of the E1α subunit of pyruvate dehyrogenase (PDH) complex, inhibition of glycolysis, retarded tumor growth, and impaired lung colonization. Enforced expression of PDK4 refractory to inhibition by miR-129-5p rescued all of these phenotypes. Targeting PDK4 by shRNA recapitulated the effects caused by miR-129-5p. miR-129-5p is transcriptionally repressed by a complex comprised of Myc, histone deacetylase 3 (HDAC3), and enhancer of zeste 2 polycomb repressive complex 2 (EZH2). Levels of miR-129-5p negatively correlated with clinical stages in human HCC. Restoring miR-129-5p expression suppressed the diethylnitrosamine (DEN)-induced hepatocarcinogenesis in mice. Thus, we concluded that miR-129-5p, which is a negative target of Myc, blocks glycolysis to retard hepatocarcinogenesis via targeting PDK4. The critical link between miR-129-5p and PDK4 in the progression of HCC suggests potential points of therapeutic intervention for this disease.

Roles and regulations of the ETS transcription factor ELF4/MEF.

Abstract: Most E26 transformation-specific (ETS) transcription factors are involved in the pathogenesis and progression of cancer. This is in part due to the roles of ETS transcription factors in basic biological processes such as growth, proliferation, and differentiation, and also because of their regulatory functions that have physiological relevance in tumorigenesis, immunity, and basal cellular homoeostasis. A member of the E74-like factor (ELF) subfamily of the ETS transcription factor family-myeloid elf-1-like factor (MEF), designated as ELF4-has been shown to be critically involved in immune response and signalling, osteogenesis, adipogenesis, cancer, and stem cell quiescence. ELF4 carries out these functions as a transcriptional activator or through interactions with its partner proteins. Mutations in ELF4 cause aberrant interactions and induce downstream processes that may lead to diseased cells. Knowing how ELF4 impinges on certain cellular processes and how it is regulated in the cells can lead to a better understanding of the physiological and pathological consequences of modulated ELF4 activity.

IRE1–RACK1 axis orchestrates ER stress preconditioning-elicited cytoprotection from ischemia/reperfusion injury in liver

Abstract: Endoplasmic reticulum (ER) stress is involved in ischemic preconditioning that protects various organs from ischemia/reperfusion (I/R) injury. We established an in vivo ER stress preconditioning model in which tunicamycin was injected into rats before hepatic I/R. The hepatic I/R injury, demonstrated by serum aminotransferase level and the ultra-structure of the liver, was alleviated by administration of tunicamycin, which induced ER stress in rat liver by activating inositol-requiring enzyme 1 (IRE1) and upregulating 78 kDa glucose-regulated protein (GRP78). The proteomic identification for IRE1 binders revealed interaction and cooperation among receptor for activated C kinase 1 (RACK1), phosphorylated AMPK, and IRE1 under ER stress conditions in a spatiotemporal manner. Furthermore, in vitro ER stress preconditioning was induced by thapsigargin and tunicamycin in L02 and HepG2 cells. Surprisingly, BCL2 was found to be phosphorylated by IRE1 under ER stress conditions to prevent apoptotic process by activation of autophagy. In conclusion, ER stress preconditioning protects against hepatic I/R injury, which is orchestrated by IRE1-RACK1 axis through the activation of BCL2. Our findings provide novel insights into the molecular pathways underlying ER stress preconditioning-elicited cytoprotective effect against hepatic I/R injury.

New insights into the structural basis of DNA recognition by HINa and HINb domains of IFI16

Abstract: Interferon gamma-inducible protein 16 (IFI16) senses DNA in the cytoplasm and the nucleus by using two tandem hematopoietic interferon-inducible nuclear (HIN) domains, HINa and HINb, through the cooperative assembly of IFI16 filaments on double-stranded DNA (dsDNA). The role of HINa in sensing DNA is not clearly understood. Here, we describe the crystal structure of the HINa domain in complex with DNA at 2.55 resolution and provide the first insight into the mode of DNA binding by the HINa domain. The structure reveals the presence of two oligosaccharide/nucleotide-binding (OB) folds with a unique DNA-binding surface. HINa uses loop L-45 of the canonical OB2 fold to bind to the DNA backbone. The dsDNA is recognized as two single strands of DNA. Interestingly, deletion of HINb compromises the ability of IFI16 to induce IFN-beta, while HINa mutants impaired in DNA binding enhance the production of IFN-beta. These results shed light on the roles of IFI16 HIN domains in DNA recognition and innate immune responses.

Prostaglandin E2 receptor EP3 regulates both adipogenesis and lipolysis in mouse white adipose tissue

Abstract: Among the four prostaglandin E2 receptors, EP3 receptor is the one most abundantly expressed in white adipose tissue (WAT). The mouse EP3 gene gives rise to three isoforms, namely EP3α, EP3β, and EP3γ, which differ only at their C-terminal tails. To date, functions of EP3 receptor and its isoforms in WAT remain incompletely characterized. In this study, we found that the expression of all EP3 isoforms were downregulated in WAT of both db/db and high-fat diet-induced obese mice. Genetic ablation of three EP3 receptor isoforms (EP3-/- mice) or EP3α and EP3γ isoforms with EP3β intact (EP3β mice) led to an obese phenotype with increased food intake, decreased motor activity, reduced insulin sensitivity, and elevated serum triglycerides. Since the differentiation of preadipocytes and mouse embryonic fibroblasts to adipocytes was markedly facilitated by either pharmacological blockade or genetic deletion/inhibition of EP3 receptor via the cAMP/PKA/PPARγ pathway, increased adipogenesis may contribute to obesity in EP3-/- and EP3β mice. Moreover, both EP3-/- and EP3β mice had increased lipolysis in WAT mainly due to the activated cAMP/PKA/hormone-sensitive lipase pathway. Taken together, our findings suggest that EP3 receptor and its α and γ isoforms are involved in both adipogenesis and lipolysis and influence food intake, serum lipid levels, and insulin sensitivity.

Structure of the TBC1D7−TSC1 complex reveals that TBC1D7 stabilizes dimerization of the TSC1 C-terminal coiled coil region

Abstract: TSC1 and TSC2 mutations account for the majority of tuberous sclerosis complex cases. The TSC1 and TSC2 proteins assemble into a complex that is stabilized by TBC1D7 through its direct interaction with the TSC1 coiled coil (CC) region. Loss of TBC1D7 is associated with intellectual disability and megalencephaly. Here, we determine the crystal structure of the complex between TBC1D7 and the C-terminal part (residues 939-992) of TSC1-CC. The structure reveals that two TSC1-CCs form a parallel homodimer, which results in the formation of two symmetric surfaces for interaction with TBC1D7. TBC1D7 employs its α4 and α5 helices to interact with the α1 helix of one TSC1 (939-992) molecule mainly through hydrophobic interactions, and simultaneously associates with the other TSC1 (939-992) molecule using the C-terminal tip of its α4 helix. Biochemical and cell biological data demonstrate that TBC1D7 indeed substantially stabilizes the homodimerization of TSC1-CC, and mutations to the critical interface residues greatly compromise this effect. Together, our data reveal the molecular mechanism underlying TBC1D7-mediated stabilization of TSC1 dimerization, and its contribution to the structural integrity of the holo-TSC complex.

Bile acids evoke placental inflammation by activating Gpbar1/NF-κB pathway in intrahepatic cholestasis of pregnancy

Abstract: Intrahepatic cholestasis of pregnancy (ICP) is a cholestatic disorder with potentially deleterious consequences for fetuses. Although a clear correlation between the elevated levels of maternal serum bile acids and deficient fetal outcome has been established in clinical practice, the underlying mechanisms remain elusive. Herein, we report that bile acids induce NF-κB pathway activation via G protein-coupled bile acid receptor 1 (Gpbar1), with consequent upregulation of inflammatory genes in trophoblasts, leading to aberrant leukocyte infiltration and inflammation in placenta. Ursodeoxycholic acid (UDCA), a drug used clinically to treat ICP, competes with other bile acids for binding with Gpbar1 and thus inhibits bile acid-induced inflammatory response in trophoblasts and improves fetal survival in pregnant rats with obstructive cholestasis. Notably, inhibition of NF-κB by andrographolide is more effective than UDCA in benefiting placentas and fetuses. Thus, anti-inflammation therapy targeting Gpbar1/NF-κB pathway could be effective in suppressing bile acid-induced inflammation and alleviating ICP-associated fetal disorders.

Regulation of Hippo signalling by p38 signalling

Abstract: The Hippo signalling pathway has a crucial role in growth control during development, and its dysregulation contributes to tumorigenesis. Recent studies uncover multiple upstream regulatory inputs into Hippo signalling, which affects phosphorylation of the transcriptional coactivator Yki/YAP/TAZ by Wts/Lats. Here we identify the p38 mitogen-activated protein kinase (MAPK) pathway as a new upstream branch of the Hippo pathway. In Drosophila, overexpression of MAPKK gene licorne (lic), or MAPKKK gene Mekk1, promotes Yki activity and induces Hippo target gene expression. Loss-of-function studies show that lic regulates Hippo signalling in ovary follicle cells and in the wing disc. Epistasis analysis indicates that Mekk1 and lic affect Hippo signalling via p38b and wts We further demonstrate that the Mekk1-Lic-p38b cascade inhibits Hippo signalling by promoting F-actin accumulation and Jub phosphorylation. In addition, p38 signalling modulates actin filaments and Hippo signalling in parallel to small GTPases Ras, Rac1, and Rho1. Lastly, we show that p38 signalling regulates Hippo signalling in mammalian cell lines. The Lic homologue MKK3 promotes nuclear localization of YAP via the actin cytoskeleton. Upregulation or downregulation of the p38 pathway regulates YAP-mediated transcription. Our work thus reveals a conserved crosstalk between the p38 MAPK pathway and the Hippo pathway in growth regulation.

Comparative population genomics reveals genetic basis underlying body size of domestic chickens

Abstract: Body size is the most important economic trait for animal production and breeding. Several hundreds of loci have been reported to be associated with growth trait and body weight in chickens. The loci are mapped to large genomic regions due to the low density and limited number of genetic markers in previous studies. Herein, we employed comparative population genomics to identify genetic basis underlying the small body size of Yuanbao chicken (a famous ornamental chicken) based on 89 whole genomes. The most significant signal was mapped to the BMP10 gene, whose expression was upregulated in the Yuanbao chicken. Overexpression of BMP10 induced a significant decrease in body length by inhibiting angiogenic vessel development in zebrafish. In addition, three other loci on chromosomes 1, 2, and 24 were also identified to be potentially involved in the development of body size. Our results provide a paradigm shift in identification of novel loci controlling body size variation, availing a fast and efficient strategy. These loci, particularly BMP10, add insights into ongoing research of the evolution of body size under artificial selection and have important implications for future chicken breeding.

The tumor suppressor ING1b is a novel corepressor for the androgen receptor and induces cellular senescence in prostate cancer cells

Abstract: The androgen receptor (AR) signaling is critical for prostate cancer (PCa) progression to the castration-resistant stage with poor clinical outcome. Altered function of AR-interacting factors may contribute to castration-resistant PCa (CRPCa). Inhibitor of growth 1 (ING1) is a tumor suppressor that regulates various cellular processes including cell proliferation. Interestingly, ING1 expression is upregulated in senescent primary human prostate cells; however, its role in AR signaling in PCa was unknown. Using a proteomic approach by surface-enhanced laser desorption ionization-mass spectrometry (SELDI-MS) combined with immunological techniques, we provide here evidence that ING1b interacts in vivo with the AR. The interaction was confirmed by co-immunoprecipitation, in vitro GST-pull-down, and quantitative intracellular colocalization analyses. Functionally, ING1b inhibits AR-responsive promoters and endogenous key AR target genes in the human PCa LNCaP cells. Conversely, ING1b knockout (KO) mouse embryonic fibroblasts (MEFs) exhibit enhanced AR activity, suggesting that the interaction with ING1b represses the AR-mediated transcription. Also, data suggest that ING1b expression is downregulated in CRPCa cells compared with androgen-dependent LNCaP cells. Interestingly, its ectopic expression induces cellular senescence and reduces cell migration in both androgen-dependent and CRPCa cells. Intriguingly, ING1b can also inhibit androgen-induced growth in LNCaP cells in a similar manner as AR antagonists. Moreover, ING1b upregulates different cell cycle inhibitors including p27(KIP1), which is a novel target for ING1b. Taken together, our findings reveal a novel corepressor function of ING1b on various AR functions, thereby inhibiting PCa cell growth.

Discovering a critical transition state from nonalcoholic hepatosteatosis to nonalcoholic steatohepatitis by lipidomics and dynamical network biomarkers

Abstract: Nonalcoholic fatty liver disease (NAFLD) is a major risk factor for type 2 diabetes and metabolic syndrome. However, accurately differentiating nonalcoholic steatohepatitis (NASH) from hepatosteatosis remains a clinical challenge. We identified a critical transition stage (termed pre-NASH) during the progression from hepatosteatosis to NASH in a mouse model of high fat-induced NAFLD, using lipidomics and a mathematical model termed dynamic network biomarkers (DNB). Different from the conventional biomarker approach based on the abundance of molecular expressions, the DNB model exploits collective fluctuations and correlations of different metabolites at a network level. We found that the correlations between the blood and liver lipid species drastically decreased after the transition from steatosis to NASH, which may account for the current difficulty in differentiating NASH from steatosis based on blood lipids. Furthermore, most DNB members in the blood circulation, especially for triacylglycerol (TAG), are also identified in the liver during the disease progression, suggesting a potential clinical application of DNB to diagnose NASH based on blood lipids. We further identified metabolic pathways responsible for this transition. Our study suggests that the transition from steatosis to NASH is not smooth and the existence of pre-NASH may be partially responsible for the current clinical limitations to diagnose NASH. If validated in humans, our study will open a new avenue to reliably diagnose pre-NASH and achieve early intervention of NAFLD.

Crumbs 3b promotes tight junctions in an ezrin-dependent manner in mammalian cells

Abstract: AMT-L is supported by the School of Biology, University of St Andrews. AMT-L, PAR and FJGM were funded by the Anonymous Trust, University of St Andrews. PAR is supported by the Melville Trust for the Care and Cure of Cancer. The mass spectrometry work was supported by the Wellcome Trust [grant number 094476/Z/10/Z], which funded the purchase of the TripleTOF 5600 mass spectrometer at the BSRC Mass Spectrometry and Proteomics Facility, University of St Andrews. The clinical study was supported by the Department of Pathology, Albert Einstein College of Medicine/ Montefiore Medical Center.

BMP4 mediates the interplay between adipogenesis and angiogenesis during expansion of subcutaneous white adipose tissue

Abstract: The expansion of subcutaneous (SC) white adipose tissue (WAT) has beneficial effects on metabolic health. Our previous work showed an increased number of bone morphogenetic protein 4 (BMP4)-activated beige adipocytes in SC WAT, indicating a potential role of BMP4 in adipocyte recruitment. It was also demonstrated that BMP4 committed multipotent mesodermal C3H10T1/2 stem cells to the adipocyte lineage ex vivo However, the mechanism by which BMP4 regulates adipogenesis in vivo has not been clarified. In this study, we found that BMP4 stimulated de novo adipogenesis in SC WAT concomitant with enhanced blood vessel formation, thus promoting adipose tissue angiogenesis. Platelet-derived growth factor receptor-β-positive (PDGFRβ(+)) multipotent stem cells within the neoangiogenic vessels were found to be adipocyte progenitors. Moreover, BMP4 downregulated PDGFRβ by stimulating the lysosome-dependent degradation, which efficiently initiated adipogenic differentiation. These results suggest how BMP4 regulates adipocyte recruitment in SC WAT, and thus promote its beneficial metabolic effects.

Yin Yang 1 promotes mTORC2-mediated AKT phosphorylation

Abstract: Yin Yang 1 (YY1) regulates both gene expression and protein modifications, and has shown a proliferative role in cancers. In this study, we demonstrate that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. YY1 expression positively correlated with AKT(S473) phosphorylation in a tissue microarray and cultured cells of breast cancer, but negatively associated with the distant metastasis-free survival of 166 breast cancer patients. YY1 promotes AKT phosphorylation at S473 through direct interaction with AKT, and the AKT-binding site is mapped to the residues G201-S226 on YY1. These residues are also involved in YY1 interaction with Mdm2, Ezh2, and E1A, and thus are designated as the oncogene protein binding (OPB) domain. YY1-promoted AKT phosphorylation relies on the OPB domain but is independent of either transcriptional activity of YY1 or the activity of phosphoinositide-3-kinases. We also determine that YY1-promoted mTORC2 access to AKT leads to its phosphorylation at S473. Importantly, a peptide based on the OPB domain blocks YY1 interaction with AKT and reduces AKT phosphorylation and cell proliferation. Thus, we demonstrate for the first time that YY1 promotes mTORC2-mediated AKT activation and disrupting YY1-AKT interaction by OPB domain-based peptide may represent a potential strategy for cancer therapy.