An Fc receptor structurally related to MHC class I antigens

Maternal immunoglobulin G transmitted to the fetus or newborn provides humoral immunity for the first weeks of mammalian life. Fc receptors on intestinal epithelial cells of the neonatal rat (FcRn) mediate the uptake of IgG from milk. Affinity-purified FcRn is resolved by SDS-PAGE into components of relative molecular masses 45,000-53,000 (p51) and about 14,000 (p14). We report the identification of the smaller component as beta 2-microglobulin. Association of beta 2-microglobulin with p51 was confirmed by crosslinking in intestinal epithelial cell brush borders. We have cloned a cDNA encoding the presumptive Fc-binding subunit, p51, and its predicted primary structure has three extracellular domains and a transmembrane region which are all homologous to the corresponding domains of class I major histocompatibility complex (MHC) antigens. This is the first time a function has been assigned to an MHC antigen-related molecule.     

The Rab8 GTPase regulates apical protein localization in intestinal cells

A number of proteins are known to be involved in apical/basolateral transport of proteins in polarized epithelial cells. The small GTP-binding protein Rab8 was thought to regulate basolateral transport in polarized kidney epithelial cells through the AP1B-complex-mediated pathway. However, the role of Rab8 (Rab8A) in cell polarity in vivo remains unknown. Here we show that Rab8 is responsible for the localization of apical proteins in intestinal epithelial cells. We found that apical peptidases and transporters localized to lysosomes in the small intestine of Rab8-deficient mice. Their mislocalization and degradation in lysosomes led to a marked reduction in the absorption rate of nutrients in the small intestine, and ultimately to death. Ultrastructurally, a shortening of apical microvilli, an increased number of enlarged lysosomes, and microvillus inclusions in the enterocytes were also observed. One microvillus inclusion disease patient who shows an identical phenotype to Rab8-deficient mice expresses a reduced amount of RAB8 (RAB8A; NM_005370). Our results demonstrate that Rab8 is necessary for the proper localization of apical proteins and the absorption and digestion of various nutrients in the small intestine.     

Ligand-induced rapid redistribution of lysosomes is temporally distinct from endosome translocation

When many ligands bind to cell-surface receptors, ligand-receptor complexes are internalized via clathrin coated pits by a process called receptor-mediated endocytosis. The cytoplasmic fate of ligands internalized within endocytic vesicles or endosomes is variable. For example, maternal immunoglobulins are transported through the cytoplasm of neonatal intestinal epithelial cells and are exocytosed at the basolateral surface. However, other ligands are degraded as a result of their delivery to the lysosomal compartment of cells. Although the translocation of endosomes to the Golgi region in the cell centre seems to be a general phenomenon presumably coupled to ligand degradation by lysosomes and endosomes and lysosomes undergo saltatory movements within the cytoplasm, the spatial control of interaction between the two structures is not understood. To address this problem we have begun to examine the spatial and temporal intracellular distribution of endosomes and lysosomes. Utilizing a new fluorescent microscopic approach, we have now been able simultaneously to visualize endosome and lysosome populations in living cells. Our results suggest that a specific relocation of lysosomes is rapidly induced upon binding of different types of ligands to the cell surface; this migration of lysosomes to the Golgi region of the cell precedes the translocation of endosomes into the same area.     

小肠粘膜细胞铁吸收机制

缺铁性贫血和运动性低铁状态在运动员中发生率高于一般人群，运动员补铁效果不理想。寻求有效的补铁措施的关键问题是要阐明小肠粘膜上皮细胞究竟如何吸收铁，然而有关铁吸收的机制一直不清楚。近几年研究发现小肠粘膜上皮细胞存在DMT1、Dcytb、Fp1和Hp等铁转运蛋白TfR-1和TfR-2在隐窝细胞和绒毛细胞基底侧膜具有相对特异性分布。本根据这些发现以及对其转运机制、转运特性和调节机制的研究进展勾画出小肠粘膜上皮细胞铁吸收过程和调节机制模型。     

Clathrin is a key regulator of basolateral polarity

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.     

氢氯噻嗪在Caco-2细胞模型中的体外转运研究

采用体外培养的人小肠上皮细胞模型Caco-2考察浓度、时间、P-糖蛋白(P-glyprotein,P-gp)抑制剂维拉帕米(verapamil)以及pH对氢氯噻嗪(hydrochlorothiazide)跨膜转运的影响.氢氯噻嗪双向转运的Papp比值即Papp B→A/Papp A→B均大于1.5.在加入P-gp抑制剂维拉帕米后,氢氯噻嗪PappA→B极显著增大,Papp B→A降低,且Papp B→A/Papp A→B从2.87下降到0.67,加入前后有极显著差异(P0.01),且氢氯噻嗪的吸收转运随pH值的降低而显著增加.结果表明,氢氯噻嗪在Caco-2细胞模型中的吸收可能存在由载体介导的主动转运,同时它还受到P-糖蛋白的外排作用.     

Plasticity of functional epithelial polarity

The fundamental characteristics that allow vectorial transport across an epithelial cell are the differential sorting and insertion of transport proteins either in the apical or the basolateral plasma membrane, and the preferential association of endocytosis and exocytosis with one or the other pole of the cell. Asymmetrical cellular structure and function, being manifestations of terminal differentiation, might be expected to be predetermined and invariant. Here we show that the polarity of transepithelial H+ transport, endocytosis and exocytosis in kidney can be reversed by environmental stimuli. The HCO3- secreting cell in the cortical collecting tubule is found to be an intercalated cell possessing a Cl-/HCO3- exchanger in the apical membrane and proton pumps in endocytic vesicles that fuse with the basolateral membrane; the H+-secreting cell in the medullary collecting tubule has these transport functions on the opposite membranes. Further, the HCO3- -secreting cell can be induced to change its functional polarity to that of the H+-secreting cell by acid-loading the animal.     

An H+-ATPase in opposite plasma membrane domains in kidney epithelial cell subpopulations

Vectorial solute transport by epithelia requires the polarized insertion of transport proteins into apical or basolateral plasmalemmal domains. In the specialized intercalated cells of the kidney collecting duct, the selective placement of an apical plasma membrane proton-pumping ATPase (H+-ATPase) and of a basolateral membrane anion-exchange protein results in transepithelial proton secretion. It is currently believed that amino-acid sequences of membrane proteins contain critical signalling regions involved in sorting these proteins to specific membrane domains. Recently, it was proposed that intercalated cells can reverse their direction of proton secretion under different acid-base conditions by redirecting proton pumps from apical to basolateral membranes, and anion exchangers from basolateral to apical membranes. But others have found that antibodies raised against the red cell anion-exchange protein (Band 3) only labelled intercalated cells at the basolateral plasma membrane, providing evidence against the model of polarity reversal. In this report, we have examined directly the distribution of proton pumps in kidney intercalated cells using specific polyclonal antibodies against subunits of a bovine kidney medullary H+-ATPase. We find that some cortical collecting duct intercalated cells have apical plasma membrane proton pumps, whereas others have basolateral pumps. This is the first direct demonstration of neighbouring epithelial cells maintaining opposite polarities of a transport protein. Thus, either subtle structural differences exist between proton pumps located at opposite poles of the cell, or factors other than protein sequence determine the polarity of H+-ATPase insertion.     

Protein kinase A regulates chloride conductance in endocytic vesicles from proximal tubule.

Regulation of ion transport by phosphorylation and G proteins occurs in several epithelial and non-epithelial cell plasma membranes1-5. It is not known whether transporters on intracellular membranes are target sites for second messengers. Here we present direct evidence that a chloride conductance in endocytic vesicles from rabbit proximal tubule is activated by phosphorylation through a cyclic AMP-dependent protein kinase. To measure chloride transport, endocytic vesicles were labelled in vivo with a Cl(-)-sensitive fluorescent indicator6-8. It was found that labelled endosomes contained an inward proton pump and a chloride conductance, but no ion-coupled chloride transport, and that the chloride conductance was regulated by protein kinase A. These results, taken together with measurements of chloride effects on ATP-dependent acidification, suggest that endosomal pH can be controlled by phosphorylation of a stilbene-sensitive conductive chloride transporter.     

日本三角涡虫肠上皮的超微结构

用透射电镜镜研究了日本三角涡虫(Dugesiajaponica)肠上皮的超微结构,结果表明:肠上皮由两类细胞构成,一类为电子密度高深染的细胞,另一类为电子密度低浅染的细胞;深染的细胞没有突向肠腔,内有大小不一的未着色和深染的囊泡;浅染的细胞游离面呈不规则样突向肠腔,也含有很多大小不一的未着色和浅染的囊泡;浅染的细胞中可...     

Activation of latent Ca2+ channels in renal epithelial cells by parathyroid hormone.

Calcium has an important role in regulating epithelial cell ion transport and is itself transported by tissues involved in the maintenance of extracellular Ca2+ homeostasis. Although the mechanism of Ca2+ entry in electrically excitable cells is well-documented little is known about it in epithelial cells. Calcium absorption in polarized epithelial cells is a two-step process in which Ca2+ enters cells across apical plasma membranes and is extruded across basolateral membranes. Efflux may be mediated by an energy-dependent Ca2(+)-ATPase or by Na+/Ca2+ exchange. We examined Ca2+ influx in single cultured cells from distal renal tubules sensitive to parathyroid hormone by measuring intracellular Ca2+. Our results demonstrate that parathyroid hormone activates dihydropyridine-sensitive channels responsible for Ca2+ entry. We also show that microtubule-dependent exocytosis stimulated by parathyroid hormone may be necessary for the insertion or activation of Ca2+ channels in these cells. Once inserted or activated, dihydropyridine-sensitive channels mediate Ca2+ entry into these Ca2(+)-transporting epithelial cells. Our results support the view that agonist-induced exocytosis may represent a general paradigm for modulation of transport in epithelial cells by delivery and incorporation of transport proteins to plasma membranes or by delivery to plasma membranes of factors regulating these proteins.     

栉孔扇贝消化系统的组织学研究

以组织学方法研究了栉孔扇贝的消化系统、栉孔扇贝消化系统由食道、胃、晶杆囊、肠、直肠及消化腺等组成,消化道管壁由粘膜层和粘膜下层组成,除食道外缺乏肌层．消化道粘膜上皮主要为纤毛柱状细胞,在食道、肠和直肠还有少量的粘液细胞．消化腺为复管状腺。腺管上皮有消化细胞和嗜碱性细胞两种类型,消化细胞内含许多囊泡和颗粒。     

小鼠胃肠道5-羟色胺免疫反应阳性肥大细胞

利用免疫组化技术和甲苯胺蓝(TB)染色技术,对小鼠胃肠道5-羟色胺免疫反应阳性细胞(5-HT+细胞)、肥大细胞(MC)和5-羟色胺免疫反应阳性肥大细胞(5-HT+MC)进行了研究.结果表明:小鼠胃肠中5-HT+细胞多分布于黏膜上皮细胞之间、腺泡上皮细胞之间和固有层内;MC多分布于黏膜层和黏膜下层结缔组织之中;小鼠胃和小肠中分别有49%和47%的MC与免疫组化染色的邻片中5-HT+细胞在位置上相对应,该部分MC为5-HT+MC;小鼠胃和小肠中分别有24%和20%的5-HT+细胞为MC.本文从形态学的角度证明了小鼠胃肠道MC可分泌5-HT,MC是消化道5-HT的来源之一.     

Dependence on pH of polarized sorting of secreted proteins

The plasma membranes of epithelial cells are divided into apical and basolateral domains. These two surfaces are characterized by markedly different protein compositions, reflecting the ability of the cell to target newly synthesized membrane proteins to specific regions of the cell surface. This targeting capability is also apparent in the polarized release of secretory products. Recent studies using canine renal tubule (MDCK) cells have suggested that distinct sets of secretory proteins are released from their apical and basolateral poles. We report experiments designed to examine secretory protein sorting by MDCK cells. We have shown that secretion of basement membrane components (laminin and heparan sulphate proteoglycan (HSPG] takes place from the basolateral cell surface and that this polarized release results from active sorting. The sorting process which mediates this polarized secretion requires an acidic intracellular compartment. MDCK cells treated with NH4Cl to raise the pH of their intracellular compartments, secrete laminin and HSPG by a default pathway which leads to their release in roughly equal quantities into the medium of both the apical and basolateral compartments.     

Fc receptor phosphorylation during receptor-mediated control of B-cell activation

It is well known that Fc receptors for IgG (FcRII) on macrophages mediate the endocytosis of antibody-antigen complexes and signal the release of inflammatory and cytotoxic agents. FcRII are also expressed at high levels on B cells where they are less involved in endocytosis than in modulating B-cell activation by membrane immunoglobulins. Although crosslinking of membrane immunoglobulins can result in B-cell differentiation and proliferation through stimulation of phospholipase C, mobilization of intracellular Ca2+, and activation of protein kinase C, crosslinking FcR with membrane immunoglobulins confers a dominant inhibitory signal that prevents or aborts activation. This form of regulation may have a role in the induction of tolerance by IgG and in controlling the B-cell repertoire by anti-idiotypes. The different functions of FcR on B cells and macrophages may reflect the fact that these cell types express closely related but distinct FcR isoforms. We have recently found that the main lymphocyte FcR isoform, FcRII-B1, is unable to mediate endocytosis by way of coated pits and coated vesicles owing to an in-frame insertion of 47 amino acids in its cytoplasmic tail. Here we show that this insert, absent from the FcRII-B2 macrophage isoform, also contains serine phosphorylation sites that may have a role in the ability of FcR to regulate B-cell activation through membrane immunoglobulins.     

Positional cloning of zebrafish ferroportin1 identifies a conserved vertebrate iron exporter

Defects in iron absorption and utilization lead to iron deficiency and overload disorders. Adult mammals absorb iron through the duodenum, whereas embryos obtain iron through placental transport. Iron uptake from the intestinal lumen through the apical surface of polarized duodenal enterocytes is mediated by the divalent metal transporter, DMTi. A second transporter has been postulated to export iron across the basolateral surface to the circulation. Here we have used positional cloning to identify the gene responsible for the hypochromic anaemia of the zebrafish mutant weissherbst. The gene, ferroportin1, encodes a multiple-transmembrane domain protein, expressed in the yolk sac, that is a candidate for the elusive iron exporter. Zebrafish ferroportin1 is required for the transport of iron from maternally derived yolk stores to the circulation and functions as an iron exporter when expressed in Xenopus oocytes. Human Ferroportin1 is found at the basal surface of placental syncytiotrophoblasts, suggesting that it also transports iron from mother to embryo. Mammalian Ferroportin1 is expressed at the basolateral surface of duodenal enterocytes and could export cellular iron into the circulation. We propose that Ferroportin1 function may be perturbed in mammalian disorders of iron deficiency or overload.     

FADD prevents RIP3-mediated epithelial cell necrosis and chronic intestinal inflammation

Intestinal immune homeostasis depends on a tightly regulated cross talk between commensal bacteria, mucosal immune cells and intestinal epithelial cells (IECs). Epithelial barrier disruption is considered to be a potential cause of inflammatory bowel disease; however, the mechanisms regulating intestinal epithelial integrity are poorly understood. Here we show that mice with IEC-specific knockout of FADD (FADD(IEC-KO)), an adaptor protein required for death-receptor-induced apoptosis, spontaneously developed epithelial cell necrosis, loss of Paneth cells, enteritis and severe erosive colitis. Genetic deficiency in RIP3, a critical regulator of programmed necrosis, prevented the development of spontaneous pathology in both the small intestine and colon of FADD(IEC-KO) mice, demonstrating that intestinal inflammation is triggered by RIP3-dependent death of FADD-deficient IECs. Epithelial-specific inhibition of CYLD, a deubiquitinase that regulates cellular necrosis, prevented colitis development in FADD(IEC-KO) but not in NEMO(IEC-KO) mice, showing that different mechanisms mediated death of colonic epithelial cells in these two models. In FADD(IEC-KO) mice, TNF deficiency ameliorated colon inflammation, whereas MYD88 deficiency and also elimination of the microbiota prevented colon inflammation, indicating that bacteria-mediated Toll-like-receptor signalling drives colitis by inducing the expression of TNF and other cytokines. However, neither CYLD, TNF or MYD88 deficiency nor elimination of the microbiota could prevent Paneth cell loss and enteritis in FADD(IEC-KO) mice, showing that different mechanisms drive RIP3-dependent necrosis of FADD-deficient IECs in the small and large bowel. Therefore, by inhibiting RIP3-mediated IEC necrosis, FADD preserves epithelial barrier integrity and antibacterial defence, maintains homeostasis and prevents chronic intestinal inflammation. Collectively, these results show that mechanisms preventing RIP3-mediated epithelial cell death are critical for the maintenance of intestinal homeostasis and indicate that programmed necrosis of IECs might be implicated in the pathogenesis of inflammatory bowel disease, in which Paneth cell and barrier defects are thought to contribute to intestinal inflammation.     

Identification of stem cells in small intestine and colon by marker gene Lgr5

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. It is currently believed that four to six crypt stem cells reside at the +4 position immediately above the Paneth cells in the small intestine; colon stem cells remain undefined. Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49) was selected from a panel of intestinal Wnt target genes for its restricted crypt expression. Here, using two knock-in alleles, we reveal exclusive expression of Lgr5 in cycling columnar cells at the crypt base. In addition, Lgr5 was expressed in rare cells in several other tissues. Using an inducible Cre knock-in allele and the Rosa26-lacZ reporter strain, lineage-tracing experiments were performed in adult mice. The Lgr5-positive crypt base columnar cell generated all epithelial lineages over a 60-day period, suggesting that it represents the stem cell of the small intestine and colon. The expression pattern of Lgr5 suggests that it marks stem cells in multiple adult tissues and cancers.     

A complementary DNA clone for a macrophage-lymphocyte Fc receptor

Macrophages, granulocytes and many lymphocytes express or secrete receptors for the Fc domain of immunoglobulins (Ig). These Fc receptors (FcRs) are heterogeneous and can be distinguished on the basis of their cellular distribution and specificities for different immunoglobulin isotypes. Although their functions are not completely understood, FcRs are known to be involved in triggering various effector cell functions and in regulating differentiation and development of B-cells. One of the best characterized is the mouse macrophage-lymphocyte receptor for IgG1 and IgG2b (ref. 5). On macrophages, this FcR mediates the endocytosis of antibody-antigen complexes via coated pits and coated vesicles, the phagocytosis of Ig-coated particles, and the release of various inflammatory and cytotoxic agents. It is possible that the receptor possesses an intrinsic ligand-activated ion channel activity responsible for some of these functions. The IgG1/IgG2b FcR has been isolated and shown to be a transmembrane glycoprotein of relative molecular mass (Mr) 47,000-60,000 (47-60 K) containing four N-linked oligosaccharide chains and a large (greater than 10K) cytoplasmic domain. It is also immunologically indistinguishable from the murine Ly-17 alloantigen which, in turn, is tightly linked to the Mls lymphocyte activation locus. Here we describe the isolation and characterisation of a complementary DNA clone encoding the whole of the IgG1/IgG2b FcR expressed by the mouse macrophage-like cell line P388D1. The receptor is a member of the immunoglobulin superfamily and, like Ly-17, maps to the distal portion of chromosome 1. cDNA probes detect one or two mRNA species in FcR+ macrophage and B-cell lines, but not in FcR- cells or a receptor-deficient variant derived from a FcR+ B-cell line. Finally, DNA hybridization analysis indicates the receptor gene is partially deleted or rearranged in the FcR- variant.