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Endocrinology 145(2):1011–1016 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2003-0652
Expression and Distribution of MAL2, an Essential Element of the Machinery for Basolateral-to-Apical Transcytosis, in Human Thyroid Epithelial Cells ´ NICA MARAZUELA, FERNANDO MARTI´N-BELMONTE, MARI´A ANGELES GARCI´A-LO ´ PEZ, MO JUAN F. ARANDA, MARI´A C. DE MARCO, AND MIGUEL A. ALONSO Departamento de Endocrinologı´a (M.M., M.A.G.-L.), Hospital de la Princesa, 28006 Madrid, Spain; and Centro de Biologı´a Molecular “Severo Ochoa” (F.M-B., J.F.A., M.C.de.M., M.A.A.), Universidad Auto´noma de Madrid and Consejo Superior de Investigaciones Cientı´ficas, 28049 Madrid, Spain Polarized transport of newly synthesized proteins to the apical surface of epithelial cells takes place by a direct pathway from the Golgi or by an indirect route involving the delivery of the protein to the basolateral surface, followed by its endocytosis and transport across the cell. The indirect pathway, named transcytosis, is also used to translocate external material across the cell. MAL, a raft-associated integral membrane protein required for the direct apical route, is known to be expressed in the thyroid epithelium. MAL2, a member of the MAL protein family, has been recently identified as an essential component of the machinery for the transcytotic route in human hepatoma cells. Herein, we have investigated the expression and distribution of MAL2 in the human thyroid. MAL2 mRNA species were detected in the thyroid. Immunohistochemical analysis of thyroid follicles indicated that, in
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HYROID EPITHELIAL CELLS are polarized cells displaying two highly specialized plasma membrane subdomains. The free surface, the apical membrane, faces the lumen of the follicle, whereas the basolateral membrane faces the extracellular material of the basement membrane. The exchange of materials between the external milieu and the body’s interior is controlled in polarized epithelia by transcellular transport across the cells and by paracellular flux through the tight junctions (1). Transcellular transport involves a specialized pathway, known as transcytosis, consisting of endocytosis of cargo (soluble molecules, macromolecules, and even entire pathogens) on one side of the epithelial barrier, its vectorial traffic across the cell in vesicular carriers, and subsequent delivery to the other side (2). In addition to translocating external material across epithelia, the transcytotic pathway is used as an indirect route for targeting newly synthesized proteins to the apical surface via the basolateral membrane (2, 3). Depending on the tissue, the indirect pathway coexists, to a greater or lesser extent, with direct routes of apical transport from the Golgi. A direct transport pathway seems to be mediated by integration of cargo protein into specialized membrane microdomains or rafts that give rise to vesicular carriers destined for the apical Abbreviation: mAb, Monoclonal antibodies. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.
contrast to MAL, which predominantly distributed to the Golgi region, MAL2 distributed to the apical membrane. Biochemical analysis in primary thyrocyte cultures indicated that MAL2 exclusively resides in raft membranes. Confocal immunofluorescence analysis of thyrocyte cultures revealed that MAL2 predominantly localized in a subapical endosome compartment that was positive for Rab11a. Alterations in MAL2 expression, distribution, and appearance were found in specific types of follicular cell-derived carcinomas. Although the role of MAL2 has not been directly addressed in this study, the simultaneous expression of MAL and MAL2 suggests that traffic to the apical membrane in thyrocytes may rely on MAL for the direct route and on MAL2 for the transcytotic pathway. (Endocrinology 145: 1011–1016, 2004)
surface (4). MAL is a nonglycosylated integral membrane protein of 17 kDa containing four hydrophobic segments (5). MAL localizes at steady-state predominantly in the apical zone of polarized epithelia (6) and continuously shuttles between the Golgi and the plasma membrane (7). Using MDCK cells whose endogenous MAL was depleted, an essential role has been demonstrated for MAL as an element of the machinery necessary for apical transport of membrane and secretory proteins by the direct route (8 –11). MAL is the founder member of a family of proteins, referred to as the MAL protein family, with 20 – 40% overall identity at the amino acid level and similar hydrophobicity profiles (12). At least some of the members of the MAL family display unusually high hydrophobicity that allowed them to be classified as proteolipid proteins (12). The MAL2 cDNA (GenBank accession no. AY007723) encodes a novel member of the MAL family identified by yeast two-hybrid screening using a TPD52L2 bait (13), corresponding to a member of the TPD52 family expressed in human breast carcinoma (14). MAL2 has recently been identified as an essential component of the machinery for basolateral-to-apical transcytosis in hepatoma HepG2 cells (15). Thyroid epithelial cells express MAL, consistent with the existence of a raft-mediated route of transport to the apical surface directly from the Golgi in these cells (6). To investigate whether, as in HepG2 cells, MAL2 could mediate the transcytotic route in epithelial thyroid cells, we have investigated the expression of MAL2 in normal thyroid. Immunohistochemical analysis of normal
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thyroid follicles revealed that MAL2 distributes at the apical membrane in a different compartment from that containing MAL. Confocal immunofluorescence and biochemical analyses of primary thyrocyte cultures indicated that this compartment is subjacent to the apical membrane and is also positive for Rab11a, a marker of apical recycling endosomes (16). It is of special note that the expression, distribution, or appearance of MAL2 was altered in specific types of thyroid tumors. The expression of MAL2 in normal thyroid and its distribution in apical recycling endosomes led us to propose that, in addition to direct transcytosis in hepatocytes, the MAL2-mediated transcytotic pathway may also operate in thyroid epithelial cells. Materials and Methods Materials Rabbit polyclonal antibodies to Rab11a were obtained from Zymed (San Francisco, CA). Rabbit antimannosidase II antibodies were kindly provided by Dr. V. Malhotra (University of California, San Diego, CA). horseradish peroxidase-conjugated secondary anti-IgG antibodies were supplied by Pierce (Rockford, IL). Fluorochrome-conjugated secondary antibodies were from Southern Biotech (Birmingham, AL). The monoclonal antibodies (mAb) 6D9 and 9D1 specific to human MAL and MAL2 have been characterized previously (6, 15).
Cell culture conditions Human thyroid follicular cells were prepared as reported previously (17). Briefly, thyroid specimens were minced with scissors and digested with 1 mg/ml collagenase (Roche, Mannheim, Germany) in DMEM for 1 h at 37 C. The digested sample was then passed through a sieve, and the cells were collected. After extensive washing, cells were seeded in 100-mm Petri dishes (1.0 ⫻ 106 cell/ml) and cultured in DMEM supplemented with 10% FBS, penicillin (50 U/ml), 10 mU/ml thryrotropin, and streptomycin (50 g/ml), at 37 C in an atmosphere of 5% CO2–95% air. After one passage under the same culture conditions, cells were used for experimental work 7 d after being seeded.
Northern blot analysis
Marazuela et al. • MAL2 in Thyroid Epithelial Cells
sively, and developed using an enhanced chemiluminescence Western blotting kit (ECL, Amersham, Little Chalfont, UK).
Confocal immunofluorescence analysis Primary thyrocytes were grown to confluency on 0.4-m pore size polycarbonate tissue culture inserts (Transwell, Costar Inc., Cambridge, MA). The integrity of the cell monolayer was monitored by measuring the transepithelial electrical resistance using the Millicell ERS apparatus (Millipore Corp.) to assure proper cell polarization. Cells were fixed in 4% paraformaldehyde for 15 min, rinsed, and treated with 10 mm glycine for 5 min to quench the aldehyde groups. The cells were then permeabilized with 0.2% Triton X-100, rinsed, and incubated with 3% BSA in PBS for 15 min. For double-label immunofluorescence analysis, cells were incubated for 1 h with the suitable primary antibodies, rinsed several times, and incubated for 1 h with the appropriate fluorescent secondary antibodies. The secondary antibodies included goat antimouse antibodies and antirabbit IgG antibodies preadsorbed to mouse serum. Controls to assess labeling specificity included incubations with control primary antibodies or omission of the primary antibodies. After extensive washing, the coverslips were mounted on slides. Images were obtained using a Radiance 2000 Confocal Laser microscope (Bio-Rad, Hercules, CA).
Immunohistochemical analysis For immunohistochemical analysis, normal thyroid tissue was obtained from thyroid glands from patients who needed thyroid biopsy during parathyroidectomy. Specimens from epithelial-derived thyroid neoplasms were received in the Pathology Department of the Hospital de la Princesa (Madrid, Spain). All studies using human samples were done in accordance with the principles set out in the declaration of Helsinki and were approved by the local institutional ethical committee. The tissue was fixed for several hours in 10% neutral buffered formalin and subjected to routine tissue processing and paraffin embedding. Five-micrometer sections were prepared, blocked with a control antibody, and then sequentially incubated with anti-MAL2 9D1 or anti-MAL 6D9 mAb and peroxidase-conjugated antimouse IgG antibodies. Sections were developed with 0.5 mg/ml 3,3⬘-diaminobenzidine tetrahydrochloride and hydrogen peroxide, and counterstained with hematoxylin.
Approximately 20 g total RNA from the appropriated tissues was denatured in 50% formamide and 2.2 m formaldehyde at 65 C, subjected to electrophoresis, and transferred to nylon membranes. RNA samples were hybridized, under standard conditions, to 32P-labeled full-length cDNA probes corresponding to MAL (5) and MAL2 (13). Blots were finally hybridized to a 0.6-kb Hinf I/BamH I cDNA fragment from the 3⬘ untranslated region of human -actin mRNA, as a loading control. Final blot washing conditions were 0.2⫻ saline sodium citrate/0.1% sodium dodecyl sulfate (1⫻ saline sodium citrate ⫽ 0.15 m NaCl, 0.015 m sodium citrate, pH 7.0) at 65 C.
Detergent extraction procedures and immunoblot analysis Lipid rafts were prepared essentially as described by Brown and Rose (18). Primary thyrocyte cultures (8.0 ⫻ 106 cells), grown in 100-mm dishes, were rinsed with PBS and lysed for 20 min in 1 ml of 25-mm Tris-HCl, pH 7.5, 150 mm NaCl, 5 mm EDTA, 1% Triton X-100 at 4 C. The lysate was brought to 40% sucrose (wt/wt) in a final vol of 4 ml and placed at the bottom of an 8-ml 5–30% linear sucrose gradient made in the same buffer without Triton X-100. Gradients were centrifuged for 18 h at 39,000 rpm at 4 C in a Beckman SW41 rotor (High Wycombe, UK). Fractions of 1 ml were harvested from the bottom of the tube, and aliquots were subjected to immunoblot analysis. Samples were then subjected to SDS-PAGE in 15% acrylamide gels under reducing conditions and transferred to Immobilon-P membranes (Millipore Corp., Bedford, MA). After blocking with 5% nonfat dry milk, 0.05% Tween 20 in PBS, blots were incubated with the indicated primary antibody. After several washings, blots were incubated for 1 h with secondary goat anti-IgG antibodies coupled to horseradish peroxidase, washed exten-
FIG. 1. Expression of the MAL2 gene in different tissues. Total RNA (⬃20 g) from the appropriate human tissues was hybridized to a full-length MAL2 cDNA probe. In parallel, a similar blot was hybridized to a full-length MAL cDNA probe and, after stripping, to an actin cDNA probe. The hybridization signal obtained with the actin cDNA probe was used as a control for the loading of similar amounts of RNA in each lane.
Marazuela et al. • MAL2 in Thyroid Epithelial Cells
Results and Discussion Expression of the MAL2 gene in the thyroid
To investigate the expression of the MAL2 gene in the thyroid, we carried out Northern blot analysis of thyroid RNA using a human MAL2 cDNA probe. RNA samples from other tissues were analyzed in parallel. Figure 1 shows that the thyroid expresses MAL2 mRNA species of the same size (2.8 kb) as those reported previously (13, 15). MAL2 gene expression was also found in stomach and at lower levels in testis and small intestine, and it was undetectable in the thymus. Consistent with our previous results (6), high levels of MAL mRNA transcripts (1.1 kb) were observed in the thyroid, thymus, and stomach; low expression in the small intestine; and undetectable expression in testis. Distribution of MAL2 in thyroid follicles
To examine the distribution of MAL2, tissue sections of normal human thyroid were subjected to immunohistochemical analysis with anti-MAL2 mAb 9D1, and the staining was compared with that obtained with the anti-MAL
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mAb 6D9. Figure 2 shows that, in agreement with the presence of MAL2 transcripts in the thyroid, positive MAL2 staining was found in thyroid epithelial cells. MAL2 distribution was restricted to the apical region of the epithelial surface. By contrast, MAL showed a supranuclear distribution at the apical part of the cell, corresponding to the Golgi area. No staining was found at the basal or lateral membranes with either anti-MAL or anti-MAL2 antibodies. Endogenous MAL2 is present in rafts in thyroid epithelial cells
To analyze the expression of MAL2, primary thyrocytes cultured as described in Materials and Methods were analyzed by flow cytometry with anti-MAL2 mAb 9D1. Figure 3A shows that approximately 60% of the thyrocytes retained expression of MAL2. To isolate fractions enriched in lipid rafts, the thyrocyte cultures were extracted with 1% Triton X-100 at 4 C, and the extracts were centrifuged to equilibrium in sucrose density gradients by using an established protocol (18). Twelve 1-ml fractions were obtained after fractionation
FIG. 2. MAL2 distribution in thyroid follicles. Thyroid tissue sections were subjected to immunohistochemical analysis with anti-MAL2 mAb 9D1 (A and B) and anti-MAL mAb 6D9 (C), and counterstained with hematoxylin to visualize nuclei. Notice the specific labeling of MAL2 in the apical membrane of thyrocytes, whereas MAL is predominantly concentrated in a supranuclear compartment. No labeling was detected for any of the proteins in the lateral or basal membrane subdomains. Original magnification, ⫻450.
FIG. 3. Identification of endogenous MAL2 in rafts in primary thyrocyte cultures. A, Primary cultures of thyrocytes (8.0 ⫻ 106 cells) were analyzed by flow cytometry with anti-MAL2 mAb 9D1 or a negative control X63 antibody. B, Primary cultures of thyrocytes were extracted with 1% Triton X-100 at 4 C and subjected to centrifugation to equilibrium in sucrose density gradients. Fractions of 1 ml were collected from the bottom of the tube. Aliquots from each fraction were subjected to SDS-PAGE and analyzed by immunoblotting with anti-MAL2 and anti-MAL antibodies as indicated. The position of glycosylated and unglycosylated MAL2 is indicated in the corresponding blot. The asterisk indicates the position of protein bands that react unspecifically with the secondary antibody used. Fractions 1– 4 are the 40% sucrose layer and contain the bulk of cellular membranes and cytosolic proteins, whereas fractions 5–12 are the 5–30% sucrose layer and contain rafts. As a control of the fractionation procedure, the distribution of the transferrin receptor (TfR), a protein known to be excluded from rafts, was determined in the different fractions of the gradient. The position of molecular weight standards is indicated.
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of the gradient from the bottom of the tube. The detergentresistant material containing the rafts floated in the gradient (fractions 5–12), whereas the bulk of cellular membranes and cytosolic proteins remained in the 40% sucrose cushion (fractions 1– 4). Aliquots from each fraction were separated by SDS-PAGE and analyzed by immunoblotting with antiMAL2 mAb 9D1 and anti-MAL mAb 6D9. Figure 3B shows that both MAL2 and MAL were exclusively detected in the floating membranes, indicating that the two proteins specifically reside in raft membranes in thyroid epithelial cells. As a control of the fractionation procedure, we observed that the transferrin receptor, a membrane protein known to be excluded from rafts, was identified only in the soluble fractions. Distribution of MAL2 in thyroid epithelial cells
To investigate the localization of MAL2 in polarized cells, we analyzed the distribution of MAL2 by confocal immunofluorescence microscopic analysis using thyrocyte cultures polarized in culture inserts. Figure 4 shows different x–y sections corresponding to horizontal planes in the most apical part of the cell, in a plane equidistant from the apical surface and the nucleus and in a plane close to the basal surface. MAL2 staining was detected in the top part of the cell, although the labeling was stronger in the medial apical plane. A subapical compartment was strongly labeled, although a diffuse vesicular pattern was also evident. The
FIG. 4. Confocal immunofluorescence analysis of the distribution of MAL2 in polarized cultures of primary thyrocytes. Thyrocytes grown to confluence in culture inserts were analyzed by confocal immunofluorescence with anti-MAL2 and antimannosidase II antibodies. The sections corresponding to the indicated horizontal planes are shown. Bar, 10 m.
FIG. 5. Colocalization of MAL2 with Rab11a in polarized thyrocytes. Thyrocytes grown to confluence in culture inserts were analyzed by confocal immunofluorescence microscopy with anti-MAL2 and anti-Rab11a antibodies. The section corresponding to the indicated subapical plane is shown. Bar, 3 m.
Marazuela et al. • MAL2 in Thyroid Epithelial Cells
distribution of MAL2 was clearly different from that of the Golgi apparatus, as indicated by double staining with mannosidase II, a marker of the Golgi middle cisterna. Finally, no significant MAL2 staining was obtained in the basal plane. To characterize the subapical compartment that concentrates most of the MAL2, double-label immunofluorescence analysis was carried out with anti-Rab11a antibodies, which preferentially labels the apical recycling compartment (16). Figure 5 shows significant colocalization of MAL2 with Rab11a in a subapical plane (arrows), suggesting that MAL2 is present in the apical recycling compartment of thyroid epithelial cells. Expression of MAL2 in thyroid carcinomas
The essential role of MAL2 in basolateral-to-apical traffic and its expression in thyroid epithelial cells imply that alterations in the expression and/or distribution of MAL2 would probably be reflected in abnormal cell function. It has been known, for a long time, that tumor formation in epithelia is frequently accompanied by disruption of normal cell polarity (19). It is therefore plausible that the alteration in the machinery for protein sorting, at least in some types of tumors, may contribute to the loss of cell polarity. In the case of MAL, we have already documented such changes in specific types of renal and thyroid carcinomas (20). In esophageal carcinomas, a clear relationship has recently been established between the progression of the tumor and the loss of MAL expression (21). To investigate whether MAL2 expression and/or distribution are altered in thyroid cancer, we have carried out an immunohistochemical analysis of different types of thyroid tumors. Figure 6 shows the distinct staining pattern of thyroid follicular cell-derived carcinomas: in all cases examined (n ⫽ 7), papillary carcinoma showed MAL2 staining similar to that of normal thyroid (Fig. 6A); the staining of the follicular carcinoma (n ⫽ 5) was localized to the apical side but had a more diffuse appearance than that in normal tissue (Fig. 6B); and MAL2 expression was not detected in anaplastic carcinoma (n ⫽ 2) (Fig. 6C). Normal staining was found in the tissue adjacent to the tumor cells in all cases (data not shown). MAL2 and MAL as elements of the integral membrane protein machinery for raft-mediated trafficking in thyroid epithelial cells
Lipid rafts have been proposed as playing an important role in the specific recruitment of proteins involved in the direct pathway of apical transport. The role of rafts in the transcytotic route has been controversial: transcytosing pIgR
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FIG. 6. MAL2 expression in thyroid tumors of follicular cells. A, Papillary carcinoma. Staining for MAL2 was found in the apical side (⫻40). B, Follicular carcinomas. A diffuse pattern of MAL2 staining was found in the apical side (⫻40). C, Anaplastic carcinoma. No staining was found (⫻40).
incorporates into rafts during movement to the apical membrane in intestinal cell explants (22) but remains excluded from that fraction in epithelial MDCK cells (23). However, recent evidence obtained in polarized hepatic cells clearly supports the involvement of rafts in the transcytotic pathway (15, 24, 25). The observations that: 1) MAL2, which is raftassociated, is required for basolateral-to-apical transcytosis in HepG2 cells; 2) transcytosing pIgR accumulation in HepG2 cells with depleted levels of MAL2 is similar to that found when lipid rafts are disrupted by cholesterol depletion (our unpublished results); and 3) the incorporation of the transcytosing pIgR into rafts, as determined by in situ extraction with Triton X-100, is prevented in cells with reduced levels of MAL2 (our unpublished results), suggest that MAL2 is involved in the egress of the transcytosing cargo from perinuclear endosomes in order for it to travel to the apical surface via a raft-dependent pathway. Therefore, lipid rafts seem to be involved in both the direct and the indirect apical routes of transport. The indirect transcytotic route constitutes a common pathway for most polarized epithelia, whereas the direct route is exclusive to certain types of epithelial cells. Thyroid epithelial cells are known to use both pathways to target proteins to the apical surface. We have found that MAL2 is expressed in the thyroid epithelium and, consistent with their respective specialized tasks, MAL2 and MAL distributed to different raft-containing subcellular compartments. Therefore, MAL and MAL2 are probably part of the raft-specific machinery that acts in thyroid epithelial cells to transport proteins to the apical surface by the direct and the indirect routes, respectively. The alterations in MAL2 expression, distribution, or appearance observed in specific types of tumors open up the possibility that the analysis of MAL2 expression could aid the characterization of thyroid cancers and the identification of incipient thyroid neoplasms. Acknowledgments We thank S. Go´ mez and C. Sa´ nchez for technical help. Received May 27, 2003. Accepted October 15, 2003. Address all correspondence and requests for reprints to: Miguel A. Alonso, Centro de Biologı´a Molecular “Severo Ochoa,” Universidad Auto´noma de Madrid, Cantoblanco, 28049 Madrid, Spain. E-mail:
[email protected]. This work was supported by grants from the Ministerio de Ciencia y Tecnologı´a (PM99-0092; BMC2003-03297), the Comunidad de Madrid (08.5/0066.1/2001), Fondo de Investigacio´n Sanitaria (01/0085-01 and -02), and Fundacio´n Eugenio Rodriguez Pascual. An institutional grant from the Fundacio´n Ramo´n Areces to Centro de Biologı´a Molecular “Severn Ochoa” is also acknowledged.
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Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving the endocrine community.
