Phospholipid flippases attenuate LPS-induced TLR4 signaling by mediating endocytic retrieval of Toll-like receptor 4

P4-ATPases are lipid flippases that catalyze the transport of phospholipids to create membrane phospholipid asymmetry and to initiate the biogenesis of transport vesicles. Here we show, for the first time, that lipid flippases are essential to dampen the inflammatory response and to mediate the endotoxin-induced endocytic retrieval of Toll-like receptor 4 (TLR4) in human macrophages. Depletion of CDC50A, the β-subunit that is crucial for the activity of multiple P4-ATPases, resulted in endotoxin-induced hypersecretion of proinflammatory cytokines, enhanced MAP kinase signaling and constitutive NF-κB activation. In addition, CDC50A-depleted THP-1 macrophages displayed reduced tolerance to endotoxin. Moreover, endotoxin-induced internalization of TLR4 was strongly reduced and coincided with impaired endosomal MyD88-independent signaling. The phenotype of CDC50A-depleted cells was also induced by separate knockdown of two P4-ATPases, namely ATP8B1 and ATP11A. We conclude that lipid flippases are novel elements of the innate immune response that are essential to attenuate the inflammatory response, possibly by mediating endotoxin-induced internalization of TLR4.     

An MD2-derived peptide promotes LPS aggregation,facilitates its internalization in THP-1 cells,and inhibits LPS-induced pro-inflammatory responses

MD2, a 160-residue accessory glycoprotein, is responsible for the recognition and binding of Gram-negative bacterial membrane component, lipopolysaccharide (LPS). Internalization of pathogen inside the mononuclear phagocytes has also been attributed to MD2 which leads to the clearance of pathogens from the host. However, not much is known about the segments in MD2 that are responsible for LPS interaction or internalization of pathogen inside the defense cells. A 16-residue stretch (MD54) from MD2 protein has been identified that possesses a short heptad repeat sequence and four cationic residues enabling it to participate in both hydrophobic and electrostatic interactions with LPS. An MD54 analog of the same size was also designed in which a leucine residue at a heptadic position was replaced with an alanine residue. MD54 but not its analog, MMD54 induced aggregation of LPS and aided in its internalization within THP-1 monocytes. Furthermore, MD54 inhibited LPS-induced nuclear translocation of NF-κB in PMA-treated THP-1 and TLR4/MD2/CD14-transfected HEK-293T cells and the production of pro-inflammatory cytokines. In addition, in in vivo experiments, MD54 showed marked protection and survival of mice against LPS-induced inflammation and death. Overall, we have identified a short peptide with heptad repeat sequence from MD2 that can cause aggregation of LPS and abet in its internalization within THP-1 cells, resulting in attenuation of LPS-induced pro-inflammatory responses in vitro and in vivo.     

The modular nature of apoptotic signaling proteins

Apoptosis, initiated by a variety of stimuli, is a physiological process that engages a well-ordered signaling cascade, eventually leading to the controlled death of the cell. The most extensively studied apoptotic stimulus is the binding of death receptors related to CD95 (Fas/Apo1) by their respective ligands. During the last years, a considerable number of proteins have been identified which act together in the receptor-proximal part of the signaling pathway. Based on localized regions of sequence similarity, it has been predicted that these proteins consist of several independently folding domains. In several cases these predictions have been confirmed by structural studies; in other cases they are at least supported by experimental data. This review focuses on the three most widespread domain families found in the apoptotic signaling proteins: the death domain, the death effector domain and the caspase recruitment domain. The recently discovered analogies between these domains, both in structure and in function, have shed some light on the overall architecture of the pathway leading from death receptor ligation to the activation of caspases and eventually to the apoptotic phenotype. Received 8 October 1998; received after revision 8 January 1999; accepted 8 January 1999     

Role of FGFRL1 and other FGF signaling proteins in early kidney development

The mammalian kidney develops from the ureteric bud and the metanephric mesenchyme. In mice, the ureteric bud invades the metanephric mesenchyme at day E10.5 and begins to branch. The tips of the ureteric bud induce the metanephric mesenchyme to condense and form the cap mesenchyme. Some cells of this cap mesenchyme undergo a mesenchymal-to-epithelial transition and differentiate into renal vesicles, which further develop into nephrons. The developing kidney expresses Fibroblast growth factor (Fgf)1, 7, 8, 9, 10, 12 and 20 and Fgf receptors Fgfr1 and Fgfr2. Fgf7 and Fgf10, mainly secreted by the metanephric mesenchyme, bind to Fgfr2b of the ureteric bud and induce branching. Fgfr1 and Fgfr2c are required for formation of the metanephric mesenchyme, however the two receptors can substitute for one another. Fgf8, secreted by renal vesicles, binds to Fgfr1 and supports survival of cells in the nascent nephrons. Fgf9 and Fgf20, expressed in the metanephric mesenchyme, are necessary to maintain survival of progenitor cells in the cortical region of the kidney. FgfrL1 is a novel member of the Fgfr family that lacks the intracellular tyrosine kinase domain. It is expressed in the ureteric bud and all nephrogenic structures. Targeted deletion of FgfrL1 leads to severe kidney dysgenesis due to the lack of renal vesicles. FgfrL1 is known to interact mainly with Fgf8. It is therefore conceivable that FgfrL1 restricts signaling of Fgf8 to the precise location of the nascent nephrons. It might also promote tight adhesion of cells in the condensed metanephric mesenchyme as required for the mesenchymal-to-epithelial transition.     

LIM domain only 4 protein promotes granulocyte colony-stimulating factor-induced signaling in neurons

Granulocyte colony-stimulating factor (GCSF) is currently in clinical trials to treat neurodegenerative diseases and stroke. Here, we tested whether LIM domain only 4 protein (LMO4), a hypoxia-inducible gene that protects neurons from ischemic injury, could modulate the neuroprotective effect of GCSF. We showed that GCSF treatment acetylates and phosphorylates Stat3, activates expression of a Stat3-dependent anti-apoptotic gene, p27, and increases neuron survival from ischemic injury. LMO4 participates in Stat3 signaling in hepatocytes and associates with histone deacetylase 2 (HDAC2) in cancer cells. In the absence of LMO4, GCSF fails to rescue neurons from ischemic insults. In wild-type neurons, inhibition of HDAC promoted Stat3 acetylation and the antiapoptotic effect of GCSF. In LMO4 null cortical neurons, expression of wild-type but not HDAC-interaction-deficient LMO4 restored GCSF-induced Stat3 acetylation and p27 expression. Thus, our results indicate that LMO4 enhances GCSF-induced Stat3 signaling in neurons, in part by sequestering HDAC.     

Effects of cytochalasin D on the actin cytoskeleton: association of neoformed actin aggregates with proteins involved in signaling and endocytosis

Cytochalasin D (CD) has been extensively used for assessing the role of the actin cytoskeleton in different biological processes. However, effects of CD have not always been consistent and CD-treated cells have been found to contain irregular spots of F-actin. By transfecting MCF-7 cells with an actin-enhanced yellow fluorescent protein fusion protein we show that, in vivo, CD induces actin aggregation de novo, while simultaneously depolymerizing preexisting actin cytoskeletal components. We also show that CD-induced actin aggregates bind the F-actin-selective drug phalloidin and associate with proteins involved in cell signaling as well as with receptors and endosomal markers (active MAP kinases, paxillin, erbB2, transferrin, Rab-5), but not with clathrin, protein kinase A, protein tyrosine phosphatase 1B, or tubulin. Thus, CD induces new sites of actin aggregation that selectively associate with several important regulatory proteins. Failure of CD to interupt a biological process may therefore not prove that the process is independent of actin aggregation.     

Zebrafish rgs4 is essential for motility and axonogenesis mediated by Akt signaling

The schizophrenia susceptibility gene, Rgs4, is one of the most intensively studied regulators of G-protein signaling members, well known to be fundamental in regulating neurotransmission. However, little is known about its role in the developing nervous system. We have isolated zebrafish rgs4 and shown that it is transcribed in the developing nervous system. Rgs4 knockdown did not affect neuron number and patterning but resulted in locomotion defects and aberrant development of axons. This was confirmed using a selective Rgs4 inhibitor, CCG-4986. Rgs4 knockdown also attenuated the level of phosphorylated-Akt1, and injection of constitutively-activated AKT1 rescued the motility defects and axonal phenotypes in the spinal cord but not in the hindbrain and trigeminal neurons. Our in vivo analysis reveals a novel role for Rgs4 in regulating axonogenesis during embryogenesis, which is mediated by another schizophrenia-associated gene, Akt1, in a region-specific manner.     

Biological functions and signaling of a transmembrane semaphorin,CD100/Sema4D

The semaphorin proteins were identified originally as axonal guidance factors functioning during neuronal development. In addition to this function, several semaphorins play diverse roles outside the nervous system. The class 4 semaphorin CD100/Sema4D, which utilizes plexin-B1 and CD72 as receptors, exerts important biological effects on a variety of cells, including the neuronal, epithelial and immune cells. Here, we review recent advances exploring the molecular mechanisms governing the biological functions of CD100/Sema4D.Received 1 July 2003; received after revision 25 July 2003; accepted 29 July 2003     

Control of infection by pyroptosis and autophagy: role of TLR and NLR

Cells can die by distinct mechanisms with particular impacts on the immune response. In addition to apoptosis and necrosis, recent studies lead to characterization of a new pro-inflammatory form of cell death, pyroptosis. TLR and NLR, central innate immune sensors, can control infections by modulating host cell survival. In addition, TLRs can promote the induction of autophagy, thus promoting delivery of infecting pathogens to the lysosomes. On the other hand, activation of some NLR members, especially NLRC4 and NAIP5, leads to the infected cell death by pyroptosis, which is accompanied by secretion of the pro-inflammatory cytokines IL-1β, IL-18, and IL-33. Data presented here illustrate how the compartmentalization of the innate immune sensors can influence the outcome of infections by controlling the fate of host cells.     

Neuronal ciliary signaling in homeostasis and disease

Primary cilia are a class of cilia that are typically solitary, immotile appendages present on nearly every mammalian cell type. Primary cilia are believed to perform specialized sensory and signaling functions that are important for normal development and cellular homeostasis. Indeed, primary cilia dysfunction is now linked to numerous human diseases and genetic disorders. Collectively, primary cilia disorders are termed as ciliopathies and present with a wide range of clinical features, including cystic kidney disease, retinal degeneration, obesity, polydactyly, anosmia, intellectual disability, and brain malformations. Although significant progress has been made in elucidating the functions of primary cilia on some cell types, the precise functions of most primary cilia remain unknown. This is particularly true for primary cilia on neurons throughout the mammalian brain. This review will introduce primary cilia and ciliary signaling pathways with a focus on neuronal cilia and their putative functions and roles in human diseases.     

Hedgehog signaling in pancreas development and disease

Since its discovery, numerous studies have shown that the Hedgehog (Hh) signaling pathway plays an instrumental role during diverse processes of cell differentiation and organ development. More recently, it has become evident that Hh signaling is not restricted to developmental events, but retains some of its activity during adult life. In mature tissues, Hh signaling has been implicated in the maintenance of stem cell niches in the brain, renewal of the gut epithelium and differentiation of hematopoietic cells. In addition to the basal function in adult tissue, deregulated signaling has been implicated in a variety of cancers, including basal cell carcinoma, glioma and small cell lung cancer. Here, we will focus on the role of Hh signaling in pancreas development and pancreatic diseases, including diabetes mellitus, chronic pancreatitis and pancreatic cancer. Received 5 August 2005; received after revision 4 November 2005; accepted 22 November 2005     

Metabolic and redox signaling in the retina

Visual perception by photoreceptors relies on the interaction of incident photons from light with a derivative of vitamin A that is covalently linked to an opsin molecule located in a special subcellular structure, the photoreceptor outer segment. The photochemical reaction produced by the photon is optimal when the opsin molecule, a seven-transmembrane protein, is embedded in a lipid bilayer of optimal fluidity. This is achieved in vertebrate photoreceptors by a high proportion of lipids made with polyunsaturated fatty acids, which have the detrimental property of being oxidized and damaged by light. Photoreceptors cannot divide, but regenerate their outer segments. This is an enormous energetic challenge that explains why photoreceptors metabolize glucose through aerobic glycolysis, as cancer cells do. Uptaken glucose produces metabolites to renew that outer segment as well as reducing power through the pentose phosphate pathway to protect photoreceptors against oxidative damage.     

Leptin signaling and circuits in puberty and fertility

Leptin is an adipocyte-derived hormone involved in a myriad of physiological process, including the control of energy balance and several neuroendocrine axes. Leptin-deficient mice and humans are obese, diabetic, and display a series of neuroendocrine and autonomic abnormalities. These individuals are infertile due to a lack of appropriate pubertal development and inadequate synthesis and secretion of gonadotropins and gonadal steroids. Leptin receptors are expressed in many organs and tissues, including those related to the control of reproductive physiology (e.g., the hypothalamus, pituitary gland, and gonads). In the last decade, it has become clear that leptin receptors located in the brain are major players in most leptin actions, including reproduction. Moreover, the recent development of molecular techniques for brain mapping and the use of genetically modified mouse models have generated crucial new findings for understanding leptin physiology and the metabolic influences on reproductive health. In the present review, we will highlight the new advances in the field, discuss the apparent contradictions, and underline the relevance of this complex physiological system to human health. We will focus our review on the hypothalamic circuitry and potential signaling pathways relevant to leptin’s effects in reproductive control, which have been identified with the use of cutting-edge technologies of molecular mapping and conditional knockouts.     

异丙酚对大鼠内毒素脑损伤STAT3表达的影响

目的 研究异丙酚对大鼠内毒素脑损伤中信号转导子与转录激活子-3( STAT3)表达的影响,探讨异丙酚在脑损伤中的保护作用及其机制.方法 健康清洁级SD大鼠72只,雌雄不限,体重220～ 250 g,随机分为3组(n=24):L组(内毒素组)和LP组(内毒素+异丙酚组)经颈内动脉注射内毒素200 μg建立大鼠内毒素脑损伤模型,C组(对照组)经颈内动脉注射等量生理盐水,LP组颈内动脉注射内毒素后即予异丙酚100 mg/kg剂量腹腔注射.3组分别于6、12、24和48h随机处死6只大鼠,取额叶皮质,检测脑组织含水量,免疫组织化学检测P-STAT3、NF-κB和iNOS表达水平的变化,Western blot法检测大鼠内毒素脑损伤后P-STAT3蛋白表达水平的变化.结果 与C组相比,L组、LP组各时间点脑组织含水量、P-STAT3、NF-κB和iNOS表达增加(P ＜0.05,P＜0.01);与L组比较,LP组各时间点脑含水量、P-STAT3、NF-κB和iNOS表达明显减少(P＜0.05,P＜0.01).结论 异丙酚可减轻大鼠内毒素性脑损伤,机制可能与抑制脑组织磷酸化STAT3、NF-κB和iNOS表达水平上调,进而减轻炎性反应有关.     

Agar electrophoresis of soluble proteins isolated from cellular fractions of CCl4-treated rat liver

Riassunto Gli autori hanno studiato, mediante elettroforesi su agar, il comportamento delle proteine solubili isolate dalle diverse frazioni cellulari di epatociti di ratto, in corso di steatosi da CCl₄.A carico delle frazioni nucleare e mitocondriale è stata osservata una diminuzione dei componenti più e meno dispersi, con un raggruppamento in zone a motilità intermedia, pari a quella delle ₁-globuline sieriche o delle albumine, accanto ad una riduzione del numero dei componenti proteici chiaramente individuabili.Analoghe modificazioni sono state rilevate a carico dei componenti glico- e lipoproteici — nella sola frazione mitocondriale.