Lipocalin-2 (24p3/neutrophil gelatinase-associated lipocalin (NGAL)) receptor is expressed in distal nephron and mediates protein endocytosis

In the kidney, bulk reabsorption of filtered proteins occurs in the proximal tubule via receptor-mediated endocytosis (RME) through the multiligand receptor complex megalin-cubilin. Other mechanisms and nephron sites for RME of proteins are unclear. Recently, the secreted protein 24p3 (lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL)), which is expressed in the distal nephron, has been identified as a sensitive biomarker of kidney damage. A high-affinity receptor for 24p3 (24p3R) that is involved in endocytotic iron delivery has also been cloned. We investigated the localization of 24p3R in rodent kidney and its role in RME of protein-metal complexes and albumin. Immunostaining of kidney tissue showed expression of 24p3R in apical membranes of distal tubules and collecting ducts, but not of proximal tubule. The differential expression of 24p3R in these nephron segments was confirmed in the respective cell lines. CHO cells transiently transfected with 24p3R or distal tubule cells internalized submicromolar concentrations of fluorescence-coupled proteins transferrin, albumin, or metallothionein (MT) as well as the toxic cadmium-MT (Cd2+(7)-MT) complex, which caused cell death. Uptake of MT or transferrin and Cd2+(7)-MT toxicity were prevented by picomolar concentrations of 24p3. An EC50 of 123±50 nM was determined for binding of MT to 24p3R by microscale thermophoresis. Hence, 24p3R binds proteins filtered by the kidney with high affinity and may contribute to RME of proteins, including 24p3, and to Cd2+(7)-MT toxicity in distal nephron segments.     

Transgenic Expression of Human Lysophosphatidic Acid Receptor LPA2 in Mouse Intestinal Epithelial Cells Induces Intestinal Dysplasia

Lysophosphatidic acid (LPA) acts on LPA₂ receptor to mediate multiple pathological effects that are associated with tumorigenesis. The absence of LPA₂ attenuates tumor progression in rodent models of colorectal cancer, but whether overexpression of LPA₂ alone can lead to malignant transformation in the intestinal tract has not been studied. In this study, we expressed human LPA₂ in intestinal epithelial cells (IECs) under control of the villin promoter. Less than 4% of F1-generation mice had germline transmission of transgenic (TG) human LPA₂; as such only 3 F1 mice out of 72 genotyped had TG expression. These TG mice appeared anemic with hematochezia and died shortly after birth. TG mice were smaller in size compared with the wild type mouse of the same age and sex. Morphological analysis showed that TG LPA₂ colon had hyper-proliferation of IECs resulting in increased colonic crypt depth. Surprisingly, TG small intestine had villus blunting and decreased IEC proliferation and dysplasia. In both intestine and colon, TG expression of LPA₂ compromised the terminal epithelial differentiation, consistent with epithelial dysplasia. Furthermore, we showed that epithelial dysplasia was observed in founder mouse intestine, correlating LPA₂ overexpression with epithelial dysplasia. The current study demonstrates that overexpression of LPA₂ alone can lead to intestinal dysplasia.     

Siderocalin/Lcn2/NGAL/24p3 Does Not Drive Apoptosis Through Gentisic Acid Mediated Iron Withdrawal in Hematopoietic Cell Lines

Siderocalin (also lipocalin 2, NGAL or 24p3) binds iron as complexes with specific siderophores, which are low molecular weight, ferric ion-specific chelators. In innate immunity, siderocalin slows the growth of infecting bacteria by sequestering bacterial ferric siderophores. Siderocalin also binds simple catechols, which can serve as siderophores in the damaged urinary tract. Siderocalin has also been proposed to alter cellular iron trafficking, for instance, driving apoptosis through iron efflux via BOCT. An endogenous siderophore composed of gentisic acid (2,5-dihydroxybenzoic acid) substituents was proposed to mediate cellular efflux. However, binding studies reported herein contradict the proposal that gentisic acid forms high-affinity ternary complexes with siderocalin and iron, or that gentisic acid can serve as an endogenous siderophore at neutral pH. We also demonstrate that siderocalin does not induce cellular iron efflux or stimulate apoptosis, questioning the role siderocalin plays in modulating iron metabolism.     

Odorant Stimulation Promotes Survival of Rodent Olfactory Receptor Neurons via PI3K/Akt Activation and Bcl-2 Expression

Olfactory stimulation activates multiple signaling cascades in order to mediate activity-driven changes in gene expression that promote neuronal survival. To date, the mechanisms involved in activity-dependent olfactory neuronal survival have yet to be fully elucidated. In the current study, we observed that olfactory sensory stimulation, which caused neuronal activation, promoted activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway and the expression of Bcl-2, which were responsible for olfactory receptor neuron (ORN) survival. We demonstrated that Bcl-2 expression increased after odorant stimulation both in vivo and in vitro. We also showed that odorant stimulation activated Akt, and that Akt activation was completely blocked by incubation with both a PI3K inhibitor ({"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002) and Akt1 small interfering RNA. Moreover, blocking the PI3K/Akt pathway diminished the odorant-induced Bcl-2 expression, as well as the effects on odorant-induced ORN survival. A temporal difference was noted between the activation of Akt1 and the expression of Bcl-2 following odorant stimulation. Blocking the PI3K/Akt pathway did not affect ORN survival in the time range prior to the increase in Bcl-2 expression, implying that these two events, activation of the PI3K pathway and Bcl-2 induction, were tightly connected to promote post-translational ORN survival. Collectively, our results indicated that olfactory activity activated PI3K/Akt, induced Bcl-2, and promoted long term ORN survival as a result.     

Isoform-Specific Regulation and Localization of the Coxsackie and Adenovirus Receptor in Human Airway Epithelia

Adenovirus is an important respiratory pathogen. Adenovirus fiber from most serotypes co-opts the Coxsackie-Adenovirus Receptor (CAR) to bind and enter cells. However, CAR is a cell adhesion molecule localized on the basolateral membrane of polarized epithelia. Separation from the lumen of the airways by tight junctions renders airway epithelia resistant to inhaled adenovirus infection. Although a role for CAR in viral spread and egress has been established, the mechanism of initial respiratory infection remains controversial. CAR exists in several protein isoforms including two transmembrane isoforms that differ only at the carboxy-terminus (CAR^Ex7 and CAR^Ex8). We found low-level expression of the CAR^Ex8 isoform in well-differentiated human airway epithelia. Surprisingly, in contrast to CAR^Ex7, CAR^Ex8 localizes to the apical membrane of epithelia where it augments adenovirus infection. Interestingly, despite sharing a similar class of PDZ-binding domain with CAR^Ex7, CAR^Ex8 differentially interacts with PICK1, PSD-95, and MAGI-1b. MAGI-1b appears to stoichiometrically regulate the degradation of CAR^Ex8 providing a potential mechanism for the apical localization of CAR^Ex8 in airway epithelial. In summary, apical localization of CAR^Ex8 may be responsible for initiation of respiratory adenoviral infections and this localization appears to be regulated by interactions with PDZ-domain containing proteins.     

Embryonic Expression of Vasoactive Intestinal Peptide (VIP) and VIP Receptor Genes

Abstract: Vasoactive intestinal peptide (VIP) exhibits pronounced effects on the growth rate of cultured mouse embryonic day (E) 9.5 embryos and acts in tissue culture as a potent glial mitogen and neuron survival factor. However, previous studies using immunohistochemistry or in situ hybridization in the rat have not revealed the presence and location of VIP or VIP mRNA in the early developing embryo CNS. Using a sensitive in situ hybridization assay with a ³³P-labeled riboprobe, we show here that the VIP gene is expressed at least as early as E11 in the mouse hindbrain. Northern blot analysis on RNA from brain dissected from mouse embryos beginning at E14 confirmed that a correct-size mRNA for VIP was present by E14 and at later time points. Expression of the VIP2 receptor gene was also detected by northern analysis in E14 mouse brains. These studies support the hypothesis that VIP produced by the embryo exerts important effects on embryonic nervous system development.     

Expression and localisation of brain-type organic cation transporter (BOCT/24p3R/LCN2R) in the normal rat hippocampus and after kainate-induced excitotoxicity

The iron siderophore binding protein lipocalin 2 (LCN2, also known as 24p3, NGAL and siderocalin) may be involved in iron homeostasis, but to date, little is known about expression of its putative receptor, brain-type organic cation transporter (BOCT, also known as BOCT1, 24p3R, NGALR and LCN2R), in the brain during neurodegeneration. The present study was carried out to elucidate the expression of LCN2 and BOCT in hippocampus after excitotoxicity induced by the glutamate analog, kainate (KA) and a possible role of LCN2 in neuronal injury. As reported previously, a rapid and sustained induction in expression of LCN2 was found in the hippocampus after intracerebroventicular injection of KA. BOCT was expressed in neurons of the saline-injected control hippocampus, and immunolabel for BOCT protein was preserved in pyramidal neurons of CA1 at 1 day post-KA injection, likely due to the delayed onset of neurodegeneration after KA injection. At 3 days and 2 weeks after KA injections, loss of immunolabel was observed due to degenerated neurons, although remaining neurons continued to express BOCT, and induction of BOCT was found in OX-42 positive microglia. This resulted in an overall decrease in BOCT mRNA and protein expression after KA treatment. Increased expression of the pro-apoptotic marker, Bim, was found in both neurons and microglia after KA injection, but TUNEL staining indicating apoptosis was found primarily in Bim-expressing neurons, but not microglia. Interaction between LCN2 and BOCT was found by DuoLink assay in cultured hippocampal neurons. Apo-LCN2 without iron caused no significant differences in neuronal Bim expression or cell survival, whereas holo-LCN2 consisting of LCN2:iron:enterochelin complex increased Bim mRNA expression and decreased neuronal survival. Together, results suggest that LCN2 and BOCT may have a role in neuronal injury.     

Gene Expression and Functional Annotation of the Human Ciliary Body Epithelia

Purpose

The ciliary body (CB) of the human eye consists of the non-pigmented (NPE) and pigmented (PE) neuro-epithelia. We investigated the gene expression of NPE and PE, to shed light on the molecular mechanisms underlying the most important functions of the CB. We also developed molecular signatures for the NPE and PE and studied possible new clues for glaucoma.

Methods

We isolated NPE and PE cells from seven healthy human donor eyes using laser dissection microscopy. Next, we performed RNA isolation, amplification, labeling and hybridization against 44×k Agilent microarrays. For microarray conformations, we used a literature study, RT-PCRs, and immunohistochemical stainings. We analyzed the gene expression data with R and with the knowledge database Ingenuity.

Results

The gene expression profiles and functional annotations of the NPE and PE were highly similar. We found that the most important functionalities of the NPE and PE were related to developmental processes, neural nature of the tissue, endocrine and metabolic signaling, and immunological functions. In total 1576 genes differed statistically significantly between NPE and PE. From these genes, at least 3 were cell-specific for the NPE and 143 for the PE. Finally, we observed high expression in the (N)PE of 35 genes previously implicated in molecular mechanisms related to glaucoma.

Conclusion

Our gene expression analysis suggested that the NPE and PE of the CB were quite similar. Nonetheless, cell-type specific differences were found. The molecular machineries of the human NPE and PE are involved in a range of neuro-endocrinological, developmental and immunological functions, and perhaps glaucoma.     

人血管内皮生长因子受体KDR胞外3区的表达与配体结合活性的鉴定

血管内皮生长因子受体(VEGFR)是血管内皮生长因子的特异性受体,VEGFR-2在介导VEGF刺激内皮细胞增殖及血管通透性等生物学活性中起重要作用.在大肠杆菌中实现可溶性的人血管内皮生长因子受体KDR胞外3区的表达,并鉴定其与配体结合的活性.采用重叠PCR的方法合成人血管内皮生长因子受体KDR胞外3区基因,将该基因与高效表达载体pET-32a重组,转化大肠杆菌Rosetta(DE3)中,表达产物依次经过CM阳离子交换树脂和镍柱亲和层析纯化.利用ELISA法和体外抑制VEGF刺激的人脐静脉内皮细胞增殖实验检测表达产物与配体结合的活性.SDS-PAGE显示,目的蛋白主要以可溶性Trx-KDR3融合蛋白表达于胞质,30℃时1 mmol/L IPTG诱导细菌5 h融合蛋白表达量约占胞质可溶性总蛋白的20%,经CM阳离子交换树脂和镍柱亲合层析纯化得到纯度为95%的产物,Western blotting鉴定是目的蛋白.ELISA和体外HUVEC细胞增殖实验显示,表达产物具有特异性结合hVEGF165的活性,且该作用呈一定的浓度依赖性.具有配体特异性结合活性的可溶性人血管内皮生长因子受体KDR胞外3区成功表达,为靶向血管抗肿瘤治疗和相关抗体的研究奠定了基础.     

人小肠三叶因子（hITF）基因在生菜中的整合与表达

用根癌土壤杆菌（Agrobacterium tumefaciens(Smith et Townsend)conn)介导的叶盘法,将人小肠三叶因子（hITF)导入生菜（Lactuca sativa L.)中,在含有除草上培养基上筛选,获得抗性植株,通过PCR和Southern印迹分析证明,hITF cDNA已整合到生菜基因组中,Western印迹分析证明hITF在生菜中的表达。ELISA检测表明,hITF在生菜新鲜叶片中的表达量为200－300ng/g,最高达700ng/g,约占总可溶性蛋白的0．1％。     

Induced Expression of Trimerized Intracellular Domains of the Human Tumor Necrosis Factor (TNF) p55 Receptor Elicits TNF Effects

The various biological activities of tumor necrosis factor (TNF) are mediated by two receptors, one of 55 kD (TNF-R55) and one of 75 kD (TNF-R75). Although the phenotypic and molecular responses elicited by TNF in different cell types are fairly well characterized, the signaling pathways leading to them are so far only partly understood. To further unravel these processes, we focused on TNF-R55, which is responsible for mediating most of the known TNF effects. Since several studies have demonstrated the importance of receptor clustering and consequently of close association of the intracellular domains for signaling, we addressed the question of whether clustering of the intracellular domains of TNF-R55 (TNF-R55i) needs to occur in structural association with the inner side of the cell membrane, where many signaling mediators are known to reside. Therefore, we investigated whether induced intracellular clustering of only TNF-R55i would be sufficient to initiate and generate a full TNF response, without the need for a full-length receptor molecule or a transmembrane region. Our results provide clear evidence that inducible forced trimerization of either TNF-R55i or only the death domain elicits an efficient TNF response, comprising activation of the nuclear factor κB, induction of interleukin-6, and cell killing.     

Brain-derived Neurotrophic Factor (BDNF) Induces Sustained Intracellular Ca2+ Elevation through the Up-regulation of Surface Transient Receptor Potential 3 (TRPC3) Channels in Rodent Microglia

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca²⁺]_i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca²⁺ elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca²⁺ elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders.     

人类免疫缺陷病毒Ⅰ型核心蛋白(p24)在大肠杆菌中的表达、纯化及鉴定

在大肠杆菌中,利用新构建的含T7g-10L RBS以及λ-PR启动子的新型原核表达载体,通过表达gag-pol基因片段,获得了具有天然序列的人类免疫缺陷病毒1型(HIV-1)核心蛋白p24的高效表达。克隆的gag-pol基因片段在其阅读框架移位区域插入了4bp碱基,其表达的病毒蛋白酶在阅读框架上与gag一致,从而实现了对gag-pol融合蛋白的有效加工,产生成熟的核心蛋白p24及其它产物。重组p24以可溶形式存在,可以被抗p24的单克隆抗体特异识别。测定的N端8个氨基酸序列与从病毒纯化的p24完全一致。在使用硫酸铵沉淀后,采用两步离子柱层析,可将重组蛋白纯化到95％以上的纯度。结果表明,纯化的p24可以作为特异性很强的试剂而用于HIV感染的诊断及病情的预后,并可用于p24的生化及结构分析。