SPLUNC1 regulates airway surface liquid volume by protecting ENaC from proteolytic cleavage

Many epithelia, including the superficial epithelia of the airways, are thought to secrete “volume sensors,” which regulate the volume of the mucosal lining fluid. The epithelial Na⁺ channel (ENaC) is often the rate limiting factor in fluid absorption, and must be cleaved by extracellular and/or intracellular proteases before it can conduct Na⁺ and absorb excess mucosal liquid, a process that can be blocked by proteases inhibitors. In the airways, airway surface liquid dilution or removal activates ENaC. Therefore, we hypothesized that endogenous proteases are membrane-anchored, whereas endogenous proteolysis inhibitors are soluble and can function as airway surface liquid volume sensors to inhibit ENaC activity. Using a proteomic approach, we identified short palate, lung, and nasal epithelial clone (SPLUNC)1 as a candidate volume sensor. Recombinant SPLUNC1 inhibited ENaC activity in both human bronchial epithelial cultures and Xenopus oocytes. Knockdown of SPLUNC1 by shRNA resulted in a failure of bronchial epithelial cultures to regulate ENaC activity and airway surface liquid volume, which was restored by adding recombinant SPLUNC1 to the airway surface liquid. Despite being able to inhibit ENaC, recombinant SPLUNC1 had little effect on extracellular serine protease activity. However, SPLUNC1 specifically bound to ENaC, preventing its cleavage and activation by serine proteases. SPLUNC1 is highly expressed in the airways, as well as in colon and kidney. Thus, we propose that SPLUNC1 is secreted onto mucosal surfaces as a soluble volume sensor whose concentration and dilution can regulate ENaC activity and mucosal volumes, including that of airway surface liquid.     

During its nuclear phase the multifunctional regulatory protein ICP0 undergoes proteolytic cleavage characteristic of polyproteins

ICP0 is a multifunctional herpes simplex virus protein known primarily as a promiscuous transactivator. In the course of productive infection, it is localized during the first 5–7 h in the nucleus and later in the cytoplasm. In the nucleus, its primary activities are to suppress the silencing of viral DNA by host proteins, activate cdk4 through recruitment of cyclin D3 to the sites of formation of replication compartments, and degrade several cellular proteins including PML and Sp100, key components of the ND10 nuclear bodies. ICP0 is not translocated to the cytoplasm in cells infected with mutants incapable of performing these tasks. We report the unexpected finding that ICP0 is cleaved into several discrete polypeptides by a proteasome-independent process. The products of this cleavage accumulate in cells infected with ICP0 mutants incapable of degrading PML and therefore are retained in the nucleus. In the second step, the products of the initial cleavage of wild-type virus-infected cells are themselves subject to proteasome-dependent degradation. The average half life of intact ICP0 during the nuclear phase is approximately 1 h. The proteasome-independent cleavage products are no longer detected at late times corresponding to the cytoplasmic phase of ICP0. The results are consistent with the hypothesis that the cleavage products of ICP0 function in topologically distinct domains during its nuclear phase.     

Crosstalk between Wnt and Notch signaling in intestinal epithelial cell fate decision

Continuous renewal of the intestinal epithelium requires coordinated regulation to maintain the balance between proliferation and differentiation of the epithelial stem cells and immature progenitor cells. Canonical Wnt signaling has long been regarded as the signaling pathway playing a central role in this epithelial cell fate determination; however, recent studies have shown that Notch signaling is also indispensable for this process. Here, we review the current concepts of how the Wnt and Notch pathways control intestinal epithelial cell fate decisions, particularly focusing on their crosstalk at both tissue and cellular levels. As several features are shared between stem cell renewal and cancer cell renewal, comprehensive understanding of how the Wnt and Notch signaling pathways cooperate and integrate in the gut epithelium has significant implications for the development of novel therapeutic modalities for intestinal neoplasia.     

胰岛素样生长因子1通过eNOS途径调节颈动脉狭窄患者内皮祖细胞功能

目的探讨颈动脉狭窄与内皮祖细胞(EPC)的相互关系及胰岛素样生长因子1(IGF-1)对EPC的调节作用及其主要机制。方法入选脑梗死伴颈动脉狭窄患者35例为颈动脉狭窄组,同时入选健康对照者11名为对照组。根据脑血管造影结果,颈动脉狭窄组又分为轻度狭窄组、中度狭窄组、重度狭窄组。测定研究对象的血清IGF-1浓度。采用密度梯度离心法分离出单个核细胞,用内皮细胞生长培养基(EGM2)培养,双染法鉴定EPC。实验分为4组:未处理组、IGF-1组、IGF-1+氮-硝基-左旋-精氨酸甲基酯(L-NAME)组、L-NAME组,使用EGM2培养细胞2~3周,分别测定各组EPC的增殖、黏附功能。结果颈动脉狭窄程度越重,形成EPC集落数量越少(P0.05),血清IGF-1浓度也越低,并且随着狭窄程度增加,EPC增殖、黏附能力降低。功能实验显示IGF-1组EPC功能明显高于未处理组(P0.01),IGF-1+L-NAME组未见明显差异,L-NAME组低于未处理组。IGF-1组细胞内皮型一氧化氮合酶(eNOS)明显高于未处理组,IGF-1+L-NAME组未见明显差异,L-NAME组低于未处理组。结论 EPC对颈动脉狭窄可能具有保护作用,IGF-1可能通过影响eNOS的合成增强EPC的功能。     

Foetal recapitulation of nutrient surplus signalling by O-GlcNAcylation and the failing heart

The development of the foetal heart is driven by increased glucose uptake and activation of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1α (HIF-1α), which drives glycolysis. In contrast, the healthy adult heart is governed by sirtuin-1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK), which promote fatty-acid oxidation and the substantial mitochondrial ATP production required for survival in a high-workload normoxic environment. During cardiac injury, the heart recapitulates the foetal signalling programme, which (although adaptive in the short term) is highly deleterious if sustained for long periods of time. Prolonged increases in glucose uptake in cardiomyocytes under stress leads to increased flux through the hexosamine biosynthesis pathway; its endproduct – uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) – functions as a critical nutrient surplus sensor. UDP-GlcNAc drives the post-translational protein modification known as O-GlcNAcylation, which rapidly and reversibly modifies thousands of intracellular proteins. Both O-GlcNAcylation and phosphorylation act at serine/threonine residues, but whereas phosphorylation is regulated by hundreds of specific kinases and phosphatases, O-GlcNAcylation is regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds or removes GlcNAc (N-acetylglucosamine), respectively, from target proteins. Recapitulation of foetal programming in heart failure (regardless of diabetes) is accompanied by marked increases in O-GlcNAcylation, both experimentally and clinically. Heightened O-GlcNAcylation in the heart leads to impaired calcium kinetics and contractile derangements, arrhythmias related to activation of voltage-gated sodium channels and Ca²⁺/calmodulin-dependent protein kinase II, mitochondrial dysfunction, and maladaptive hypertrophy, microvascular dysfunction, fibrosis and cardiomyopathy. These deleterious effects can be prevented by suppression of O-GlcNAcylation, which can be achieved experimentally by upregulation of AMPK and SIRT1 or by pharmacological inhibition of OGT or stimulation of OGA. The effects of sodium–glucose cotransporter 2 (SGLT2) inhibitors on the heart are accompanied by reduced O-GlcNAcylation, and their cytoprotective effects are reportedly abrogated if their action to suppress O-GlcNAcylation is blocked. Such an action may represent one of the many mechanisms by which enhanced AMPK and SIRT1 signalling following SGLT2 inhibition leads to cardiovascular benefits. These observations, taken collectively, suggest that UDP-GlcNAc functions as a critical nutrient surplus sensor (which acting in concert with mTOR and HIF-1α) can promote the development of cardiomyopathy.     

肺癌与甲状腺转录因子-1的研究进展

肺癌是目前全世界发病率最高的恶性肿瘤,其病死率也居于前列.因此,对肺癌的早期诊断及早期治疗对于降低肺癌患者的病死率以及改善患者生存质量至关重要.传统的肺癌诊断方法包括影像学检查以及病理学检查,现在仍然被临床所采用.但鉴于以上检查手段的敏感性、特异性、适用度等方面存在一定局限,近年来有关肺癌早期诊断、治疗及预后的分子标志物的研究愈来愈受到关注.本文就甲状腺转录因子-1的生物作用及其在肺癌诊断、治疗及预后方面的研究进展作一综述.     

Crosstalk between the alpha2beta1 integrin and c-met/HGF-R regulates innate immunity

Data from several investigators suggest that the alpha2beta1 integrin, a receptor for collagens, laminins, decorin, E-cadherin, matrix metalloproteinase-1, endorepellin, and several viruses, is required for innate immunity and regulation of autoimmune/allergic disorders. We demonstrated that the innate immune response to Listeria monocytogenes required alpha2beta1 integrin expression by peritoneal mast cells (PMCs). Ligation of the alpha2beta1 integrin by C1q contained in immune complexes comprised of Listeria and antibody was required for PMC activation in vitro and in vivo. However, ligation of the alpha2beta1 integrin alone was insufficient to activate cytokine secretion, suggesting that one or more additional signals emanating from a coreceptor were required for PMC activation. Here, we demonstrate that C1q, but neither other complement proteins nor FcRgamma, is required for early innate immune response to Listeria. The binding of Listeria's Internalin B (InlB) to hepatocyte growth factor receptor (HGF-R)/c-met provides the costimulatory function required for PMC activation. Either HGF or Listeria InlB bound to c-met and either C1q or type I collagen bound to alpha2beta1 integrin stimulates PMC activation. These findings suggest that crosstalk between c-met and the alpha2beta1 integrin may contribute to mast-cell activation in autoimmune and inflammatory disorders.     

Evidence that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in thalassaemia

Ultrastructural immunocytochemical studies were performed on sections of bone marrow from three patients with β-thalassaemia major and two patients with haemoglobin H (HbH) disease. Some sections were reacted with either a polyclonal or a monoclonal anti-human-ubiquitin antibody and the reaction visualized using a gold-labelled secondary antibody. The inclusions of precipitated globin chains found within the erythropoietic cells of all five patients reacted much more strongly than the surrounding inclusion-free cytoplasm with both of the anti-ubiquitin antibodies, indicating that the precipitated globin chains were ubiquitinated. A non-specific reaction between the anti-ubiquitin antibodies and the inclusions was excluded by demonstrating that various other antibodies, including a polyclonal anti-human cathepsin D antibody, did not react with the inclusions. The data suggest that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in α- and β-thalassaemia.     

Three-amino-acid-loop-extension homeodomain factor Meis3 regulates cell survival via PDK1

Three-amino-acid-loop-extension (TALE) homeodomain proteins including Meis and Pbx families are generally recognized for their roles in growth and differentiation during vertebrate embryogenesis and tumorigenesis. Whereas genetic studies indicate that Pbx1 regulates the development and function of insulin-producing pancreatic β-cells, the role of Meis family members in β-cells is still unknown. Here we show that Meis3 is abundantly expressed in pancreatic islets and β-cells and that it regulates β-cell survival. We further identify the 3-phosphoinositide-dependent protein kinase 1 (PDK1), a well-known kinase involved in the PI3K-Akt signaling pathway, as a direct Meis3 target, which mediates its role in β-cell survival. This regulatory module appears to function broadly as we also identify Meis3 regulation of cell survival and PDK1 expression in ovarian carcinoma cells, suggesting a unique function for Meis3 beyond the traditional roles for TALE homeodomain factors during embryogenesis.     

TBC1D24 regulates neuronal migration and maturation through modulation of the ARF6-dependent pathway

Alterations in the formation of brain networks are associated with several neurodevelopmental disorders. Mutations in TBC1 domain family member 24 (TBC1D24) are responsible for syndromes that combine cortical malformations, intellectual disability, and epilepsy, but the function of TBC1D24 in the brain remains unknown. We report here that in utero TBC1D24 knockdown in the rat developing neocortex affects the multipolar-bipolar transition of neurons leading to delayed radial migration. Furthermore, we find that TBC1D24-knockdown neurons display an abnormal maturation and retain immature morphofunctional properties. TBC1D24 interacts with ADP ribosylation factor (ARF)6, a small GTPase crucial for membrane trafficking. We show that in vivo, overexpression of the dominant-negative form of ARF6 rescues the neuronal migration and dendritic outgrowth defects induced by TBC1D24 knockdown, suggesting that TBC1D24 prevents ARF6 activation. Overall, our findings demonstrate an essential role of TBC1D24 in neuronal migration and maturation and highlight the physiological relevance of the ARF6-dependent membrane-trafficking pathway in brain development.The mammalian cerebral cortex is a multilayered structure derived from cells of the neural tube (1). Cortical projection neurons are generated from proliferating progenitor cells located in the ventricular zone (VZ) adjacent to the lateral ventricle (2). After their final mitotic division, the majority of neurons migrate radially, along radial glia fibers, from the VZ toward the pial surface, where each successive generation passes one another and settles in an inside-out pattern within the cortical plate (CP) (3, 4). When neurons reach their final destination, they stop migrating and order themselves into specific “architectonic” patterns, according to a complex signaling pathway, guiding cells to the correct location in the brain (5). Overall, these steps lead to the formation of a six-layered cortex that plays a key role in cognitive processes. It is therefore not surprising that disruption of early cortical development causes severe neurological conditions usually featuring intellectual disabilities (IDs) and epilepsy, which may be associated with brain malformations (6).Over the past decades, advances in our understanding of the genetics of ID and epilepsy have revolutionized the diagnostic and the clinical approach to these disorders, and an increasing number of pathogenic gene mutations have been identified. TBC1D24 is a novel epilepsy-related gene mutated in different autosomal recessive forms of early-onset epilepsy, which can be accompanied by variable degrees of ID (7–11). The TBC1D24 gene encodes a protein of 553 aa that contains a Tre2/Bub2/Cdc16 (TBC) domain, shared by Rab GTPase-activating proteins (Rab-GAPs) and a TLDc domain of unknown function (8). The TBC1D24 protein is expressed in the brain and interacts with the ADP ribosylation factor (ARF) 6, a small GTP-binding protein implicated in membrane exchange between the plasma membrane and the endocytic compartments (8, 12). In the brain, ARF6 is known to play various roles including regulation of neurite outgrowth (13, 14) and maintenance of apical adhesion of neural progenitors in the developing neocortex (15). Interestingly, pathogenic mutations in TBC1D24 affect its binding to ARF6 and result in a severe impairment of neuronal development (7, 8, 10). Conversely, overexpression of TBC1D24 increases neuronal differentiation in vitro (7, 8). These findings, along with the subtle cortical malformation observed in some patients carrying a TBC1D24 mutation (7, 16), suggest that TBC1D24 is likely to be involved in brain development and maturation.To investigate TBC1D24 function in the developing neocortex, we down-regulated its expression in neuronal progenitor cells of the embryonic rat brain by in utero electroporation (IUE). We found that silencing of TBC1D24 delays radial migration and morphological maturation of pyramidal neurons. In addition, we showed that concomitant expression of an inactive form of ARF6, ARF6^T27N, was able to rescue the TBC1D24-knockdown phenotype in vivo. Furthermore, overexpression of the constitutively active ARF6^Q67L mutant results in a significant delay of neuronal migration. Overall, our findings suggest that TBC1D24, by regulating ARF6 activity in postmitotic neurons, controls radial migration and neurite outgrowth during cortical development.     

Central leptin regulates total ceramide content and sterol regulatory element binding protein-1C proteolytic maturation in rat white adipose tissue

Obesity and type 2 diabetes are associated with insulin and leptin resistance, and increased ceramide contents in target tissues. Because the adipose tissue has become a central focus in these diseases, and leptin-induced increases in insulin sensitivity may be related to effects of leptin on lipid metabolism, we investigated herein whether central leptin was able to regulate total ceramide levels and the expression of enzymes involved in ceramide metabolism in rat white adipose tissue (WAT). After 7 d central leptin treatment, the total content of ceramides was analyzed by quantitative shotgun lipidomics mass spectrometry. The effects of leptin on the expression of several enzymes of the sphingolipid metabolism, sterol regulatory element binding protein (SREBP)-1c, and insulin-induced gene 1 (INSIG-1) in this tissue were studied. Total ceramide levels were also determined after surgical WAT denervation. Central leptin infusion significantly decreased both total ceramide content and the long-chain fatty acid ceramide species in WAT. Concomitant with these results, leptin decreased the mRNA levels of enzymes involved in de novo ceramide synthesis (SPT-1, LASS2, LASS4) and ceramide production from sphingomyelin (SMPD-1/2). The mRNA levels of enzymes of ceramide degradation (Asah1/2) and utilization (sphingomyelin synthase, ceramide kinase, glycosyl-ceramide synthase, GM3 synthase) were also down-regulated. Ceramide-lowering effects of central leptin were prevented by local autonomic nervous system denervation of WAT. Finally, central leptin treatment markedly increased INSIG-1 mRNA expression and impaired SREBP-1c activation in epididymal WAT. These observations indicate that in vivo central leptin, acting through the autonomic nervous system, regulates total ceramide levels and SREBP-1c proteolytic maturation in WAT, probably contributing to improve the overall insulin sensitivity.     

RNAi screening reveals requirement for host cell secretory pathway in infection by diverse families of negative-strand RNA viruses

Negative-strand (NS) RNA viruses comprise many pathogens that cause serious diseases in humans and animals. Despite their clinical importance, little is known about the host factors required for their infection. Using vesicular stomatitis virus (VSV), a prototypic NS RNA virus in the family Rhabdoviridae, we conducted a human genome-wide siRNA screen and identified 72 host genes required for viral infection. Many of these identified genes were also required for infection by two other NS RNA viruses, the lymphocytic choriomeningitis virus of the Arenaviridae family and human parainfluenza virus type 3 of the Paramyxoviridae family. Genes affecting different stages of VSV infection, such as entry/uncoating, gene expression, and assembly/release, were identified. Depletion of the proteins of the coatomer complex I or its upstream effectors ARF1 or GBF1 led to detection of reduced levels of VSV RNA. Coatomer complex I was also required for infection of lymphocytic choriomeningitis virus and human parainfluenza virus type 3. These results highlight the evolutionarily conserved requirements for gene expression of diverse families of NS RNA viruses and demonstrate the involvement of host cell secretory pathway in the process.     

The RNA-binding protein HuD regulates neuronal cell identity and maturation

Neural Hu proteins (HuB/C/D) are RNA-binding proteins that have been shown to induce neuronal differentiation activity when overexpressed in immature neural progenitor cells or undifferentiated neuronal tumors. Newly generated HuD-deficient mice exhibited a transient impaired-cranial-nerve-development phenotype at an early embryonic stage. Adult HuD-deficient mice exhibited an abnormal hind-limb reflex and poor rotarod performance. Analysis of neurosphere formation revealed that the number and self-renewal capacity of the neural stem/progenitor cells were increased in HuD-deficient mice. HuD-deficient primary neurospheres also generated a smaller number of neurons. Cohort analysis of the cellular proliferative activity by using BrdUrd and iododeoxuridine labeling revealed that the number of differentiating quiescent cells in the embryonic cerebral wall was decreased. Long-term administration of BrdUrd revealed that the number of slowly dividing stem cells in the adult subventricular zone was increased in the HuD-deficient mice. Taken together, the results suggest that HuD is required at multiple points during neuronal development, including negative regulation of proliferative activity and neuronal cell-fate acquisition of neural stem/progenitor cells.