Expression of MAL2, an Integral Protein Component of the Machinery for Basolateral-to-Apical Transcytosis, in Human Epithelia
Departmento de Endocrinología (MM,MAGL) and Departamento de Patología (AA,MA), Hospital de la Princesa, and Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid and Consejo Superior de Investigaciones Científicas (CdM,MAA), Cantoblanco, Madrid, Spain
Correspondence to: Miguel A. Alonso, Centro de Biología Molecular "Severo Ochoa," Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain. E-mail: maalonso{at}cbm.uam.es
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Summary |
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Key Words: epithelial cell sorting machinery transcytosis MAL protein family rafts carcinoma
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Introduction |
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A direct apical transport pathway appears to be mediated by integration of cargo protein into specialized glycolipid- and cholesterol-enriched membrane microdomains or rafts that subsequently originate vesicular carriers destined for the apical surface (Simons and WandingerNess 1990). MAL is an integral, raft-associated membrane protein with a demonstrated essential role as an element of the machinery for transport of apical proteins through the direct route in MDCK cells (Puertollano et al. 1999
; Cheong et al. 1999
; MartínBelmonte et al. 2000
,2001
). The range of human epithelia that use MAL-mediated pathways of transport has recently been characterized by an immunohistochemical (IHC) survey of normal tissues (Marazuela et al. 2003
).
MAL is the founder member of the MAL family which also includes MAL2, BENE and other unedited proteins (Pérez et al. 1997). Whereas the function of BENE remains to be elucidated (de Marco et al. 2001
), MAL2 has been demonstrated to be an essential component of the machinery for basolateral-to-apical transcytosis in hepatoma HepG2 cells (de Marco et al. 2002
). HepG2 cells with reduced levels of MAL2 are unable to transport to the apical (canalicular) surface the polymeric immunoglobulin receptor and CD59, which are representatives of single transmembrane and glycosylphosphatidylinositol-anchored proteins, respectively. In this study, using an already characterized monoclonal antibody (MAb) specific for human MAL2 (de Marco et al. 2002
), we have analyzed by IHC the distribution of MAL2 in a wide variety of normal human tissues, with special emphasis on different types of epithelia, and in specific types of renal tumors.
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Materials and Methods |
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Transfection and Immunoblotting Analyis
The DNA constructs expressing the human proteins MAL2, MAL, and BENE tagged at their NH2 terminus with the 9E10 c-Myc epitope were described previously (MartínBelmonte et al. 1998; de Marco et al. 2001
,2002
). Transient transfection of COS-7 cells with these constructs was carried out by electroporation using Electro Cell Manipulator 600 equipment (BTX; San Diego, CA). For immunoblotting analysis, cells were lysed 24 hr after transfection and the extracts were subjected to SDS-PAGE in 15% acrylamide gels under reducing conditions and transferred to Immobilon-P membranes (Millipore; Bedford, MA). After blocking with 5% (w/v) non-fat dry milk, 0.05% (v/v) Tween-20 in PBS, blots were incubated with the indicated primary antibodies. After several washings, blots were incubated for 1 hr with the appropriate goat anti-IgG antibodies coupled to horseradish peroxidase (Pierce; Rockford, IL), washed extensively, and developed using an enhanced chemiluminescence Western blotting kit (Amersham; Poole, UK).
IHC Analysis of Human Samples
Normal adult tissue samples were received in the Pathology Department of the Hospital de la Princesa (Madrid, Spain) as routine specimens obtained at surgery. Specimens from epithelium-derived renal and thyroid neoplasms were also studied. These tumors included the main varieties of renal cell carcinomas: clear cell renal carcinoma (n=8), papillary renal cell carcinoma (n=4), chromophobe cell-type carcinoma (n=5), sarcomatoid renal cell carcinoma (n=2), and oncocytoma (n=6). These cases were defined according to morphology assessed using hematoxylin-stained sections and, in selected cases, immunostaining (Murphy et al. 1994).
All samples were fixed for several hours in 10% neutral buffered formalin and subjected to routine tissue processing and paraffin embedding. Sections 5 µm thick were prepared from paraffin-embedded tissues and mounted on poly-L-lysine-coated glass microslides. Antigen retrieval was accomplished by subjecting deparaffinized sections to pressure-cooker unmasking for 60 sec in 200 mM citrate buffer, pH 6.0. The tissue was then blocked with a 1:20 dilution of normal rabbit serum in 10 mM Tris-HCl saline buffer, pH 7.6, as previously described (Marazuela et al. 1995). The sections were sequentially incubated with a 1:50 dilution of a mouse ascites stock of anti-MAL2 9D1 MAb and peroxidase-conjugated rabbit anti-mouse IgG (DAKO; Glostrup, Denmark). Each incubation was followed by three washes with Tris-buffered saline. Sections were then developed with GrahamKarnovsky medium containing 0.5 mg/ml of 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical; St Louis, MO) and hydrogen peroxide. Sections were counterstained with Carazzi's hematoxylin, dehydrated, and mounted by routine methods. At least four coded samples from each tissue were examined by two independent observers.
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Results |
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Evaluation of the gastrointestinal tract included examination of esophagus, stomach, ileum, colon, liver, and pancreas. The gastrointestinal epithelium was positive for MAL2 at all sites examined. In the esophagus, the stratified squamous, non-keratinized epithelium was strongly positive (Figure 5). The staining was more pronounced in the basal layer (Figure 5). When the stratified epithelium changed to the gastric, mucus-secreting simple columnar epithelium of the stomach there was positive staining confined to the apical portion of the surface mucosa cell (Figure 6). In addition, parietal cells, responsible for the secretion of hydrochloric acid and intrinsic factor, were highly immunoreactive (Figure 6). In the small intestine, staining of the cells of the villi showed a characteristic pattern of reactivity with strong supranuclear granular positivity but no staining at the basal or lateral membranes (Figures 7A and 7B). This distribution corresponds to the location of the Golgi apparatus. In addition, secretory caliciform cells were strongly positive (Figure 7C). Throughout the gastrointestinal tract, lymphocytes from Peyer's patches, the smooth muscle of the muscularis mucosa, and the striated muscle of the muscle coat did not stain with the anti-MAL2 antibody. The mucous epithelium of the large intestine had a similar pattern of MAL2 expression, with apical staining, to that of the small intestine (Figure 7D).
In normal liver, hepatocytes showed granular cytoplasmatic staining for MAL2 (Figure 8A). Bile duct epithelium and biliary canaliculi were strongly positive (data not shown; and Figure 8B). Sinusoidal lining cells and Kupffer cells were negative. Granular positivity was found in the supranuclear area of acinic cells in the pancreas (Figure 9A). Pancreatic ducts were strongly stained (Figure 9A). In addition, sporadic cells were stained in the islets of Langerhans (Figure 9B).
Multiple sites of the genitourinary tract expressed the MAL2 molecule. In the kidney, MAL2 was widespread in specific parts of the kidney glomeruli and tubuli (Figure 10A) . In the cortex, the epithelial side of the glomerular loops showed linear staining that suggested podocyte staining (Figure 10B). Endothelial cells of the glomeruli were negative (Figure 10B). MAL2 was expressed in the distal convoluted tubules but not in the proximal tubules (Figure 10C). Staining was more pronounced in some cells of the tubules than in others (Figure 10C). Intense labeling was also observed in the collecting tubules of the medulla (our unpublished results).
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In the lung, the ciliated columnar epithelium of bronchi and bronchioles were positive (Figure 12A). Staining was confined to the apical aspect of the cells. In the alveoli, cells lining the alveolar walls were positive (Figure 12B). In addition, large cells with the appearance of type 2 pneumocytes stained strongly with anti-MAL2 antibody (Figure 12B).
In the endocrine glands, the epithelium of the prostate glands showed intense reactivity in its apical aspect (Figure 13). In the testis, Leydig cells were strongly positive (Figure 14). In addition, faint staining was found in Sertoli cells but not in spermatogenic cells (Figure 14). In the adrenal gland, the medulla showed intense granular staining (Figure 15). Although all the layers of the cortex showed staining, it was stronger in the zona reticularis (Figure 15).
The distinct types of renal cell carcinoma examined showed differential staining pattern. Renal oncocytomas showed none (Figure 16) . Although the majority of renal clear cell carcinoma showed no staining with the MAL2 antibody (Figure 17), intense focal staining was found in some of the tumors analyzed (Figure 18). Conversely, cells from chromophobe carcinomas showed intense diffuse staining, which was more pronounced on the apical side and also in some cells compared to others (Figure 19). Papillary renal cell carcinomas showed a staining similar to that of normal kidney, whereby it was more pronounced in some cells of the tubules than in others (Figure 20). Granular cell-type tumors showed intense granular cytoplasmic staining (Figure 21). Sarcomatoid carcinoma of kidney was negative for MAL2 expression (our unpublished data).
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Discussion |
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Simultaneous expression of both MAL2 and MAL was found in the epithelium of the esophagus, small and large intestine, pancreas, prostate, bronchi and trachea, and thyroid and adrenal glands. Among these epithelia are absorptive cells (e.g., enterocytes) as well as many different types of specialized secretory cells either organized in discrete clusters (e.g., endocrine cells in the pancreas), grouped in an endocrine gland (e.g., prostate), or interspersed with other cells in glands (e.g., parietal cells in the stomach). Because of its demonstrated role in apical secretion of soluble proteins (MartínBelmonte et al. 2001), in addition to transporting specific membrane proteins, the expression of MAL might be related to the secretory activity of these cell types. The MAL2-mediated pathway might be involved in these cells in the targeting of membrane proteins to the apical surface by the indirect route and in the basolateral-to apical transcellular transport of extracellular material.
An interesting scenario arises in the liver, in which all the hepatocytes are positive for MAL2 expression but only those localized in the centrilobular area express MAL. This reflects heterogeneity in the use of the direct pathway by hepatocytes related to the distance to the terminal hepatic venule, which defines the center of the centrilobular area. This differential expression of MAL is probably related to specialized tasks of centrilobular hepatocytes, such as centripetal transport from the portal space to the hepatic venule.
As examples of differences in the use of the MAL and MAL2 routes of transport in related epithelial cell types, it is worth considering a few selected cases. For example, the columnar epithelium lining the proximal convoluted tubules was negative for MAL2 and MAL expression, whereas the cuboidal epithelium of the distal convoluted tubules was positive. This difference might be related to the specific function of these two epithelia. The proximal tubule is specialized in reabsorption of components of the glomerular filtrate, whereas distal tubules control saline and acidbase balances in the urine. An interesting case is that of the cuboidal endothelium of the HEVs, which express both MAL2 and MAL, whereas the flattened endothelial cells of normal blood vessels are negative for both. Although both types of endothelia have a common role lining blood vessels and regulating blood coagulation, HEVs in lymphoid organs are specialized as the main site for constitutive extravasation during lymphocyte recircularization. The specific expression of MAL2 and MAL suggests that membrane trafficking in HEVs has additional transport requirements from those of normal endothelial cells. Another interesting comparison is that of type 1 and type 2 pneumocytes, the main cellular components of the alveoli. The flat type 1 pneumocytes, which are involved in gas exchange, do not express detectable levels of MAL although they are positive for MAL2 expression. In contrast, round type 2 pneumocytes, which secrete surfactant, a complex mixture of lipid, carbohydrates, and proteins that protects the alveolar surface, express both MAL and MAL2. This expression pattern is consistent with the idea that MAL expression might be related to surfactant secretion by type 2 pneumocytes, while MAL2 would be in charge of the indirect apical route in both types of pneumocyte.
In the case of nonepithelial cells it is worth emphasizing that T-lymphocytes express MAL but not MAL2, whereas the opposite is true in peripheral neurons and follicular dendritic cells. Mast cells were positive for both MAL2 and MAL expression. Although these cell types do not polarize segregating apical and basolateral surfaces, they are polarized cells and the expression of MAL2 and/or MAL suggests the existence in these cells of transport pathways reminiscent of the direct or indirect routes of polarized epithelia.
Applications of the Anti-MAL2 MAb 9D1
The essential role of MAL2 in raft-mediated traffic (de Marco et al. 2002) and its specific expression in polarized epithelial cell types, secretory cells, neurons, dendritic cells, and mast cells imply that alterations in the expression and/or distribution of MAL2 would probably be reflected in the abnormal functioning of the cells. In the case of MAL, we have already documented such changes in specific types of renal and thyroid carcinomas (Marazuela et al. 2003
). In mouse esophageal carcinomas there is a clear relationship between tumor progression and the loss of MAL expression (Mimori et al. 2003
). It is of particular note that ectopic expression of MAL in esophagus tumor cells prevented tumor formation and led to death of the cells by apoptosis (Mimori et al. 2003
). Therefore, MAL acts as a tumor suppressor, at least in some specific types of tumors. It is also striking that the expression and/or distribution of MAL2 were altered in specific types of renal carcinoma. Oncocytomas, which were the only type of benign renal tumor analyzed, did not express MAL2. In the particular case of clear cell carcinomas, the most common form (7080%) of renal cancers, we found heterogeneity in the expression of MAL2, which was absent in the majority of the cases. This might indicate a relationship between the grade of tumor and the expression of MAL2. The availability of the anti-human MAL2 MAb used in this study and its use in both paraffin-embedded section and cryosections may well allow the expression of MAL2 to be used as a novel tool for tumor characterization.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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