Expression of MAL2, an integral protein component of the machinery for basolateral-to-apical transcytosis, in human epithelia.

MAL2, an integral membrane protein of the MAL family, is an essential component of the machinery necessary for the indirect transcytotic route of apical transport in human hepatoma HepG2 cells. To characterize the range of human epithelia that use MAL2-mediated pathways of transport, we carried out an immunohistochemical survey of normal tissues using a monoclonal antibody specific to the MAL2 protein. MAL2 expression was detected in specific types of normal epithelial cells throughout the respiratory system, the gastrointestinal and genitourinary tracts, in exocrine and endocrine glands, and in hepatocytes. Many different types of specialized secretory cells, either organized in discrete clusters (e.g., endocrine cells in the pancreas) or in endocrine glands (e.g., prostate), were also positive for MAL2. In addition to epithelial cells, peripheral neurons, mast cells, and dendritic cells were found to express MAL2. For comparison with normal epithelial tissue, different types of renal carcinoma were also analyzed, revealing alterations in MAL2 expression/distribution dependent on the particular histological type of the tumor. Our results allow the prediction of the existence of MAL2-based trafficking pathways in specific cell types and suggest applications of the anti-MAL2 antibody for the characterization of neoplastic tissue.     

Expression of MAL and MAL2, two elements of the protein machinery for raft-mediated transport, in normal and neoplastic human tissue

Polarized transport of lipids and proteins to the apical and basolateral membrane subdomains is essential for the functioning of epithelial cells. Apical transport is mediated by a direct route from the Golgi and an indirect route, referred to as transcytosis, involving the transport of the protein to the basolateral membrane followed by its internalization and subsequent transcellular transport to the apical subdomain. MAL and MAL2 have been demonstrated to be essential components of the machinery for the direct and indirect routes, respectively. Herein, we review the range of expression of MAL and MAL2 in normal human tissue and compare it with that of neoplastic tissue. Our analysis provides insight into the potential use of MAL- and MAL2-mediated pathways in many types of epithelial cells as well as in nonepithelial cells. In addition, the specific alterations in MAL and/or MAL2 expression observed in specific types of carcinoma provides a basis to understand the loss of the polarized phenotype that frequently accompanies the neoplastic transformation process. This points out potential applications of MAL and MAL2 as markers for tumor characterization.     

MAL2, a novel raft protein of the MAL family,is an essential component of the machinery for transcytosis in hepatoma HepG2 cells

Transcytosis is used alone (e.g., hepatoma HepG2 cells) or in combination with a direct pathway from the Golgi (e.g., epithelial MDCK cells) as an indirect route for targeting proteins to the apical surface. The raft-associated MAL protein is an essential element of the machinery for the direct route in MDCK cells. Herein, we present the functional characterization of MAL2, a member of the MAL protein family, in polarized HepG2 cells. MAL2 resided selectively in rafts and is predominantly distributed in a compartment localized beneath the subapical F-actin cytoskeleton. MAL2 greatly colocalized in subapical endosome structures with transcytosing molecules en route to the apical surface. Depletion of endogenous MAL2 drastically blocked transcytotic transport of exogenous polymeric immunoglobulin receptor and endogenous glycosylphosphatidylinositol-anchored protein CD59 to the apical membrane. MAL2 depletion did not affect the internalization of these molecules but produced their accumulation in perinuclear endosome elements that were accessible to transferrin. Normal transcytosis persisted in cells that expressed exogenous MAL2 designed to resist the depletion treatment. MAL2 is therefore essential for transcytosis in HepG2 cells.     

The MAL proteolipid is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin in Madin-Darby canine kidney cells

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.     

Broad immunocytochemical localization of the formylpeptide receptor in human organs, tissues, and cells

The formylpeptide receptor (FPR), previously found only on polymorphonuclear leukocytes and monocytes/macrophages, responds to both synthetic N-formyl oligopeptides and those produced by bacteria. The cDNA for human FPR has been cloned and a rabbit polyclonal antiserum directed against a synthetic 11-amino-acid peptide corresponding to the deduced carboxy-terminus has been produced. We have now extensively characterized and used the antibody to detect FPR on normal human tissues and cell types. The receptor antigen is present on some epithelial cells, especially those with a secretory function, and on some endocrine cells, e.g., follicular cells of the thyroid and cortical cells of the adrenal. Liver hepatocytes and Kupffer cells are positive. Smooth muscle and endothelial cells are also generally positive. In the brain and spinal cord, the neurons of the motor, sensory, and cerebellar systems, and those of the parasympathetic and sympathetic systems stain positively. These data suggest that the putative endogenous agonist for FPR or an antigenically similar receptor reacts with cellular targets in the neuromuscular, vascular, endocrine, and immune systems.     

MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells.

The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.     

Immunohistochemical evidence for the overexpression of protein kinase C in proliferative diseases of human thyroid.

We report immunohistochemical evidence for the overexpression of protein kinase C in various proliferative diseases of human thyroid. Immunohistochemical characterization of various surgically removed thyroid tissues, viz., cancer tissues: papillary carcinoma and follicular carcinoma; adenoma tissues: tubular, trabecular and colloid adenomas; adenomatous goiter; and normal thyroid was done using the monospecific monoclonal antibodies MC-1a, MC-2a and MC-3a, each of which is specific for types I, II and III isozymes of protein kinase C, respectively. For protein kinase C type II, a remarkable difference in staining intensity was noted between the cancerous and normal tissues. The cytoplasm of papillary and follicular carcinoma cells stained more intensely than that of normal thyroid cells. In the benign tumor and adenomatous goiter tissues, stronger staining was noted in the papilliform-proliferating portion and cubic epithelial cells. In the normal thyroid tissues, epithelial cells of greater height were more strongly stained than simple squamous epithelial cells. These results indicated that protein kinase C type II isozyme is expressed in larger amounts in cancerous and proliferative tissues of the human thyroid.     

Characterization of anti-TIMP-1 monoclonal antibodies for immunohistochemical localization in formalin-fixed, paraffin-embedded tissue.

The aim of this study was to evaluate seven anti-TIMP-1 (tissue inhibitor of metalloproteinase-1) monoclonal antibodies by immunohistochemical (IHC) staining of formalin-fixed, paraffin-embedded (FFPE) tissue. Detection of the TIMP-1 protein was studied by IHC in FFPE human archival normal and neoplastic samples. Indirect IHC technique was used, and the seven antibodies (clones VT1, VT2, VT4, VT5, VT6, VT7, and VT8) were tested in various concentrations using different pretreatment protocols. All seven VT antibodies specifically immunostained the cytoplasm of islets of Langerhans cells in normal pancreas, epithelial cells of hyperplastic prostate, tumor cells of medullary thyroid carcinoma, and fibroblast-like cells of malignant melanoma. Specificity of the anti-TIMP-1 antibodies was confirmed by several controls, e.g., Western blotting on proteins extracted from FFPE tissue showed that the VT7 antibody reacted specifically with a protein band of approximately 28 kDa, corresponding to the molecular mass of TIMP-1. However, sensitivity varied with the different antibodies. Use of heat-induced epitope retrieval (HIER) and the VT7 clone applied at low concentrations demonstrated more intense immunoreactivity with the TIMP-1-positive cell types compared to the other six clones. Furthermore, when tested on a range of normal and neoplastic endocrine tissues, the VT7 clone demonstrated immunoreactivity with all neuroendocrine cell types. In conclusion, all seven antibodies detected TIMP-1 protein in various normal and neoplastic FFPE tissues, but one clone, VT7, was superior for IHC staining of TIMP-1 in FFPE tissue sections when using HIER.     

The MAL proteolipid is necessary for the overall apical delivery of membrane proteins in the polarized epithelial Madin-Darby canine kidney and fischer rat thyroid cell lines

The MAL proteolipid has been recently demonstrated as being necessary for correct apical sorting of the transmembrane influenza virus hemagglutinin (HA) in Madin-Darby canine kidney (MDCK) cells. The fact that, in contrast to MDCK cells, Fischer rat thyroid (FRT) cells target the majority of glycosylphosphatidylinositol (GPI)-anchored proteins to the basolateral membrane provides us with the opportunity to determine the role of MAL in apical transport of membrane proteins under conditions in which the majority of GPI-anchored proteins are (MDCK cells) or are not (FRT cells) targeted to the apical surface. Using an antisense oligonucleotide-based strategy to deplete endogenous MAL, we have observed that correct transport of apical transmembrane proteins associated (HA) or not (exogenous neurotrophin receptor and endogenous dipeptidyl peptidase IV) with lipid rafts, as well as that of the bulk of endogenous apical membrane, takes place in FRT cells by a pathway that requires normal MAL levels. Even transport of placental alkaline phosphatase, a GPI-anchored protein that is targeted apically in FRT cells, was dependent on normal MAL levels. Similarly, in addition to the reported effect of MAL on HA transport, depletion of MAL in MDCK cells caused a dramatic reduction in the apical delivery of the GPI-anchored gD1-DAF protein, neurotrophin receptor, and the bulk of membrane proteins. These results suggest that MAL is necessary for the overall apical transport of membrane proteins in polarized MDCK and FRT cells.     

Analysis of ABCC6 (MRP6) in normal human tissues

To determine the tissue distribution of the ABC transporter ABCC6 in normal human tissues, we analyzed tissue arrays for the presence of ABCC6 mRNA by in situ hybridization and ABCC6 protein by immunohistochemistry using the polyclonal antibody HB-6. We detected ABCC6 mRNA and protein in various epithelial cells of exocrine and endocrine tissues, such as acinar cells in the pancreas, mucosal cells of the intestine and follicular epithelial cells of the thyroid. We obtained a very strong immunostaining for enteroendocrine G cells in the stomach. In addition, ABCC6 mRNA and protein were present in most neurons of the brain, in alveolar macrophages in the lungs and lymphocytes in the lymph node. Immunohistochemisty using the monoclonal antibody M₆II-31 confirmed the widespread tissue distribution of ABCC6. The physiological substrate(s) of ABCC6 are yet unknown, but we suggest that ABCC6 fulfills multiple functions in different tissues. The strong immunostaining for ABCC6 in G cells suggests that it plays an important role in these endocrine cells.     

MAL, an Integral Element of the Apical Sorting Machinery, Is an Itinerant Protein That Cycles between the Trans-Golgi Network and the Plasma Membrane

The MAL proteolipid is a nonglycosylated integral membrane protein found in glycolipid-enriched membrane microdomains. In polarized epithelial Madin-Darby canine kidney cells, MAL is necessary for normal apical transport and accurate sorting of the influenza virus hemagglutinin. MAL is thus part of the integral machinery for glycolipid-enriched membrane-mediated apical transport. At steady state, MAL is predominantly located in perinuclear vesicles that probably arise from the trans-Golgi network (TGN). To act on membrane traffic and to prevent their accumulation in the target compartment, integral membrane elements of the protein-sorting machinery should be itinerant proteins that cycle between the donor and target compartments. To establish whether MAL is an itinerant protein, we engineered the last extracellular loop of MAL by insertion of sequences containing the FLAG epitope or with sequences containing residues that became O-glycosylated within the cells or that displayed biotinylatable groups. The ectopic expression of these modified MAL proteins allowed us to investigate the surface expression of MAL and its movement through different compartments after internalization with the use of a combination of assays, including surface biotinylation, surface binding of anti-FLAG antibodies, neuraminidase sensitivity, and drug treatments. Immunofluorescence and flow cytometric analyses indicated that, in addition to its Golgi localization, MAL was also expressed on the cell surface, from which it was rapidly internalized. This retrieval implies transport through the endosomal pathway and requires endosomal acidification, because it can be inhibited by drugs such as chloroquine, monensin, and NH(4)Cl. Resialylation experiments of surface MAL treated with neuraminidase indicated that approximately 30% of the internalized MAL molecules were delivered to the TGN, probably to start a new cycle of cargo transport. Together, these observations suggest that, as predicted for integral membrane members of the late protein transport machinery, MAL is an itinerant protein cycling between the TGN and the plasma membrane.     

Immunohistochemical detection of secretoneurin, a novel neuropeptide endoproteolytically processed from secretogranin II, in normal human endocrine and neuronal tissues

Summary An antiserum raised against a synthetic peptide derived from the primary amino sequence of rat secretogranin II (chromogranin C) was used for immunological (quantitative radioimmunoassay analysis) and immunohistochemical studies of normal human endocrine and nervous tissues. This antibody recognized a novel and biologically active neuropeptide which was coined as secretoneurin. In endocrine tissues, secretoneurin was mainly co-localized with chromogranin A and B with some exceptions (e.g., parathyroid gland). Secretoneurin was demonstrated immunohistochemically in the adrenal medulla, thyroid C cells, TSH- and FSH/LH-produting cells of the anterior pituitary, A and B cells of pancreatic islets, in endocrine cells of the gastrointestinal tract and the bronchial mucosa, and the prostate. Immunoreactivity determined by radioimmunoassay analysis revealed high secretoneurin levels in the anterior and posterior pituitary and lower levels in pancreatic and thyroid tissue. A strong secretoneurin immunoreactivity was also found in ganglion cells of the submucdsal and myenteric plexus of the gastrointestinal tract, and in ganglionic cells of dorsal root ganglia, peripheral nerves, and ganglion cells of the adrenal medulla. Thus, secretoneurin may serve as a useful marker of gangliocytic/neuronal differentiation.     

Immunocytochemistry of cell surface heparan sulfate proteoglycan in mouse tissues. A light and electron microscopic study

The core protein of the proteoglycan at the cell surface of NMuMG mouse mammary epithelial cells bears both heparan and chondroitin sulfate chains and is recognized by the monoclonal antibody 281-2. Using this antibody and the peroxidase-antiperoxidase staining technique in adult mouse tissues, we found that the antibody recognizes the antigen in a highly restricted distribution, staining a variety of epithelial cells but no cells derived from embryonic mesoderm or neural crest. The antibody fails to stain any stromal (mesenchymal) or neuronal cells, with the exception of plasma cells and Leydig cells. Squamous and transitional epithelia stain intensely over their entire surfaces, whereas cuboidal and columnar epithelia stain moderately and only at the lateral surface of the basal cells. Within squamous and transitional epithelial tissues that undergo physiological regeneration (e.g., epidermis), the most superficial and differentiated cell types fail to stain. Within glandular and branched epithelia (e.g., pancreas), the secretory alveolar cells fail to stain. When evaluated by electron microscopy, granular deposits of stain are seen on the plasma membrane, especially on lateral surfaces, but none are noted within the cells or the basement membrane. These results indicate that in adult tissues the core protein of this heparan sulfate-rich proteoglycan is expressed almost exclusively at epithelial cell surfaces. Expression appears to be lost as the cells become either mature or highly differentiated.     

Immunohistochemical localization of a thyroid hormone-binding protein (p55) in human tissues

We have localized p55, a thyroid hormone-binding protein found in the endoplasmic reticulum in cultured cells, in samples of normal human and monkey tissues, using a monoclonal antibody with cryostat sections and immunoperoxidase histochemistry. Large amounts of p55 were found in many tissues, generally corresponding to the amount of endoplasmic reticulum contained in each cell type. Intense localization of p55 was found in cells of the anterior and intermediate pituitary lobes, in epithelial cells of thyroid follicles, in the glandular epithelium of mammary gland, in hepatocytes, in Paneth cells and Brunner's glands in duodenum, in acinar cells of pancreas, in adrenal cortical cells, and in scattered interstitial fibroblastic cells in many tissues. These results suggest a potential role for thyroid hormone and p55 in regulating protein synthesis or secretion in multiple organs.     

Progressive segregation of unmyelinated axons in peripheral nerves, myelin alterations in the CNS, and cyst formation in the kidneys of myelin and lymphocyte protein-overexpressing mice

Myelin and lymphocyte protein (MAL) is a putative tetraspan proteolipid that is highly expressed by Schwann cells and oligodendrocytes as a component of compact myelin. Outside of the nervous system, MAL is found in apical membranes of epithelial cells, mainly in the kidney and stomach. Because MAL is associated with glycosphingolipids, it is thought to be involved in the organization, transport, and maintenance of glycosphingolipid-enriched membrane microdomains. In this report, we describe the generation and analysis of transgenic mice with increased MAL gene dosage. Immunohistochemical analysis revealed that the localization of MAL overexpression in the transgenic animals corresponded closely to the MAL expression pattern observed in wildtype animals, indicating correct spatial regulation of the transgene. Phenotypically, MAL overexpression led to progressive dissociation of unmyelinated axons from bundles in the PNS, a tendency to hypomyelination and aberrant myelin formation in the CNS, and the formation of large cysts in the tubular region of the kidney. Thus, increased expression of MAL appears to be deleterious to membranous structures in the affected tissues, indicating a requirement for tight control of endogenous MAL expression in Schwann cells, oligodendrocytes, and kidney epithelial cells.     

An Intact Dilysine-like Motif in the Carboxyl Terminus of MAL Is Required for Normal Apical Transport of the Influenza Virus Hemagglutinin Cargo Protein in Epithelial Madin-Darby Canine Kidney Cells

The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine -3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.     

Widespread expression of zinc transporter ZnT (SLC30) family members in mouse endocrine cells

Zinc is abundant in most endocrine cell types, and plays a pivotal role in the synthesis and secretion of many hormones. Recent studies have demonstrated the expression of numerous zinc transporter (ZnT) family members in the pancreas, thyroid, and adrenal glands, suggesting a role for ZnTs in regulating cellular zinc homeostasis in endocrine cells. However, the cellular distribution of ZnTs in the endocrine organs has not been well established. In the present study, the mRNA expression level, cellular distribution of ZnTs as well as liable zinc ions were examined in the mouse pituitary, adrenal glands, thyroid, and pancreas. In general, ZnT1-10 mRNA was expressed to various degrees in the detected endocrine organs, with no detectable ZnT10 mRNA in the pancreas. In the anterior pituitary, both the acidophilic and basophilic cells were immunopositive to ZnT1-5, 7, 8, except for ZnT10. In the adrenal cortex, the immunoreactivity of all the tested ZnTs, including ZnT1-5, 7, 8, 10, was observed in the zona fasciculata, and some ZnTs were detected in the zona glomerulosa, zona reticularis, and the adrenal medulla. Both the follicle epithelial cells and parafollicular cells in the thyroid gland were immunostained with ZnT1-5, 7, 8, but not ZnT10. In the endocrine pancreas, the immunoreactivity of tested ZnTs was observed to various degrees except for ZnT10 in the cytoplasm of islet cells. Furthermore, autometallographic staining showed that liable zinc was observed in the endocrine cells, such as the adrenal cortical cells, thyroid follicle epithelial cells, and the pancreatic islet cells. All together, the wide distribution of liable zinc and the phenomenon that numerous ZnT family members are partially overlapped in a subset of endocrine cells suggest an important role for the ZnT family in controlling cellular zinc levels and subsequently regulating the synthesis and secretion of hormones in the endocrine system.