Expression of MAL2,an Integral Protein Component of the Machinery for Basolateral-to-Apical Transcytosis,in Human Epithelia

Abstract

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.

Keywords

epithelial cell sorting machinery transcytosis MAL protein family rafts carcinoma

Apical transport of proteins takes place in epithelial cells by two different routes, referred to as the direct and the indirect pathways. Newly synthesized apical proteins relying on the direct route are packaged after their passage through the Golgi in vesicular carriers destined for the apical surface. In contrast, proteins transported by the indirect route are first targeted to the basolateral surface and then endocytosed and transported across the cell to the apical surface by the transcytotic pathway. All epithelia appear to use the indirect pathway, whereas the direct pathway is used to a greater or lesser extent depending on the tissue in question. Hepatocytes and hepatocyte-related cell lines (such as hepatoma HepG2 cells) mostly rely on the indirect pathway for apical transport, whereas other epithelia and most epithelial cell lines (e.g., renal MDCK cells or enterocytic Caco-2 cells) use both pathways to various degrees.

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 Wandinger-Ness 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; Martín-Belmonte 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.

Materials and Methods

Antibodies

The mouse hybridoma that produces MAb 9E10 to the c-Myc epitope was purchased from the American Type Culture Collection (Bethesda, MD) and used to produce MAb 9E10. The MAbs 9D1, 6D9, and 5B1 to human MAL2 (de Marco et al. 2002), MAL (Martín-Belmonte et al. 1998), and BENE (de Marco et al. 2001), respectively, were described previously.

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 (Martín-Belmonte 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).

Figure 1

(A) Schematic model of the predicted structure of MAL2, MAL, and BENE, with indication of the glycosylated loop of MAL2 and the peptides selected for the preparation of antibodies to each protein. (B) Protein extracts from control COS-7 cells or COS-7 cells transiently expressing tagged forms of MAL2 (19 kD), MAL (17 kD), or BENE (17 kD) were subjected to immunoblotting analysis with the antibodies 9D1, 6D9, and 5B1 generated to the indicated peptides of MAL2, MAL, and BENE. Note that, as described previously (de Marco et al. 2002), glycosylation of MAL2 in COS-7 cells is inefficient. The blot was finally analyzed with antibodies to the c-Myc tag.

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 Graham-Karnovsky 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.

Table 1

Summary of the distribution of MAL2 in different human tissues

Tissue/Organ	Strong expression	No expression
Common structures	Neurons	Endothelial cells
	Mast cells	Fibroblasts
		Muscle cells
Skin	Apical keratinizing epithelium	Basal keratinizing epithelium
	Sebaceous glands	Keratinocytes
		Hair follicles
		Melanocytes
Esophagus	Stratified squamous epithelium	Muscle cells
		Submucosa
Stomach	Parietal cells	Muscle cells
	Chief cells	Submucosa
	Surface mucous cells
Small intestine	Crypt cells	Muscle cells
	Paneth cells	Submucosa
	Enterocytes with microvilli	Lymphocytes (Peyer's patches)
Large intestine	Mucous cells	Muscle cells
		Submucosa
Liver	Hepatocytes	Kupffer cells
	Biliary canaliculi	Endothelium
	Intrahepatic ductal epithelium
Pancreas	Acinar cells
	Ductal cells
	Endocrine cells
Kidney	Glomerulus	Proximal convoluted tubules
	Distal convoluted tubules
	Loop of Henle
	Collecting tubules
Prostate	Ductal and acinar cells
Lymph node and tonsil	Dendritic cells	Lymphocytes
	HEV endothelium	Sinusoidal cells
		Macrophages
		Other endothelia
Thymus	Epithelial cells	Thymocytes
	Hassall's corpuscles
Bronchi and trachea	Respiratory epithelium
	Goblet cells
Lung	Alveolar lining cells
	Type 2 pneumocytes
	Mucous cells
Testis	Leydig cells	Germinal cells
	Sertoli cells
Adrenal gland	Medullary cells
	Zona reticularis
	Zona glomerulosa and fasciculata

Results

The predicted structure of the MAL2, MAL, and BENE proteins with indication of the peptide sequences used to prepare the corresponding MAb is depicted schematically in Figure 1A. The generation and characterization of the anti-MAL2 MAb 9D1 used for the immunohistochemical IHC analysis described in this study has been reported previously (de Marco et al. 2002). To demonstrate the lack of crossreactivity of this antibody with other members of the MAL protein family (Pérez et al. 1997), cell extracts from control untransfected COS-7 cells or COS-7 cells transiently expressing c-Myc-tagged forms of MAL2, MAL, and BENE were subjected to immunoblotting analysis with anti-MAL2 MAb 9D1, anti-MAL MAb 6D9 (Martín-Belmonte et al. 1998), and anti-BENE MAb 5B1 (de Marco et al. 2001). The blot was finally analyzed with anti-tag antibodies to ensure that the exogenous proteins were expressed at similar levels. Figure 1B shows the specific detection of each protein with its respective antibody and the absence of crossreactivity of the antibodies used, as would be expected because the antibodies were generated to peptides not conserved across the MAL protein family. The anti-BENE antibody has poor staining characteristics, whereas the anti-MAL2 and anti-MAL2 antibodies gave clear, specific, and reproducible staining on human tissues.

Figure 2

Neurons are strongly stained (arrow). Original magnification ×100.

Figure 3

Mast cells show granular staining (arrow). Original magnification ×100.

Figure 4

Skin. Sebaceous glands are stained. Original magnification ×40.

Figure 5

Esophagus. The stratified squamous epithelium is strongly positive. Original magnification ×20. Staining is more pronounced in the basal layer.

Figure 6

Stomach. The simple columnar epithelium of the gastric mucosa shows positive staining confined to the apical surface (arrow). Parietal cells are strongly positive (arrowheads). Original magnification ×20.

Figure 7

Intestine. (A) Staining is found in brush border enterocytes. Original magnification ×40. (B) At higher magnification, intense supranuclear granular staining is observed (arrows). (C) Secretory caliciform cells are also strongly positive (arrows). (D) Enterocytes from the large intestine also show apical staining (arrow). Original magnification ×100.

Figure 8

Liver. (A) Granular staining is found in hepatocytes. (B) Strong reactivity is found in biliary canaliculi (arrows). Original magnification ×100.

Figure 9

Pancreas. (A) Acinic cells (arrowheads) and pancreatic ducts (arrows) are strongly stained. Original magnification ×40. (B) Scattered cells in the islets of Langerhans (arrows) are stained with anti-MAL2 MAb. Original magnification ×20.

Because the function of MAL2 has been already established (de Marco et al. 2002), we considered it of interest to investigate the expression and distribution of MAL2 in different human tissues using the anti-MAL2 MAb 9D1. As the use of paraffin-embedded or unfixed frozen tissues in our IHC analysis gave similar patterns (our unpublished results), only the results with the former are shown. Common features observed during our analysis of human tissue specimens were the expression of MAL2 in specific epithelia, as well as in exocrine and endocrine glands, and the absence of detectable MAL2 expression in lymphocytes, fibroblasts, endothelial cells, and skeletal and smooth muscle (summarized in Table 1). In addition, mast cells (Figure 2) and the neurons of peripheral nerves (Figure 3) also showed strong immunostaining with the anti-MAL2 MAb.

Sections of skin, including keratinizing squamous epithelium, dermis with adnexa, and subcutaneous fibroadipose tissue, were examined. The apical third of the keratinizing squamous epithelium was stained (our unpublished results). All other layers of squamous epithelium as well as subcutaneous fibroadipose tissue were negative (our unpublished results). Reactivity was found in sebaceous glands (Figure 4).

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).

Figure 10

Kidney. (A) In the cortex, the glomeruli (arrows) and distal convoluted tubules are highly stained (arrowhead). Original magnification ×20. (B) The epithelial side of capillary loops shows a linear pattern of staining that suggests expression of MAL2 in podocytes (arrow). Original magnification ×100. (C) Distal convoluted tubules are highly stained in the apical border (arrow). Original magnification ×40.

Figure 11

Lymph node. (A) Staining is confined to follicular dendritic cells (arrowhead). Although no staining was found in other endothelial cells, specific staining is detected in high endothelial venules (arrow). Original magnification ×40. (B) Thymus. Staining is found in epithelial cells and Hassall's corpuscles. Original magnification ×100.

Figure 12

(A) Bronchi. Ciliated columnar epithelium of the bronchi is positive, with an apical pattern. Original magnification ×40. (B) Lung. Alveolar lining cells (arrowhead) and in type 2 pneumocytes are positive (arrows). Original magnification ×100.

Figure 13

Prostate. The epithelium of the prostate gland shows intense granular staining for MAL2. Original magnification ×40.

Figure 14

Testis. Strong granular positivity is found in secretory Leydig cells (arrowheads), while faint staining is detected in Sertoli cells (arrow). Germinal cells are negative. Original magnification ×100.

Figure 15

Adrenal gland. Secretory cells of the adrenal medulla show intense granular staining (arrowheads). All layers of the adrenal cortex stain positively, most strongly in the zona reticularis (arrows). Original magnification ×40.

The hematopoietic tissues analyzed included thymus, lymph node, and tonsil. In normal lymph node and tonsil there were similar staining patterns. Staining was confined to follicular dendritic cells (Figure 11A). In contrast to the lack of expression of the MAL2 antigen in normal endothelial cells, endothelial cells in high endothelial venules (HEVS) were strongly positive, with a characteristic apical distribution (Figure 11A). Lymphocytes were negative (Figure 11A). In the thymus, MAL2 expression was found on epithelial cells and Hassall's corpuscles (Figure 11B).

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).

Discussion

Expression of Machinery for Direct and Indirect Routes of Apical Transport in Human Epithelial Cells

The demonstrated role of MAL (Cheong et al. 1999; Puertollano et al. 1999; Martín-Belmonte et al. 2000) and MAL2 (de Marco et al. 2002) as machinery for the direct and indirect routes of apical transport, respectively, and the information provided by our IHC surveys of MAL and MAL2 expression in different tissues (compiled in Table 1) allow us to predict the potential use of the MAL- and MAL2-mediated transport pathways by specific types of epithelial cells, if we may assume that their expression is indicative of the functioning of the corresponding route. The differential expression of MAL2 and MAL in a particular epithelium is probably related to some of the specialized functions carried out by that epithelium either directly (e.g., secreting hormones) or indirectly, by transporting specific membrane receptors.

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 (Martín-Belmonte 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.

Figure 16

Renal oncocytoma shows no staining. Normal staining is found in adjacent normal tubules (arrows). Original magnification ×40.

Figure 17

Clear cell carcinoma. No staining is found in renal cell carcinoma cells. Normal staining is found in the adjacent normal kidney cells (arrow). Original magnification ×40.

Figure 18

Clear cell carcinoma. Intense staining is found in tumor cells. Original magnification ×20.

Figure 19

Chromophobe carcinoma. Diffuse pronounced staining is found in all cells, with a variable staining pattern between individual cells. Original magnification ×40.

Figure 20

Papillary renal carcinoma. Staining is more pronounced on the apical side, with more pronounced staining in some cells (arrows). Original magnification ×20.

Figure 21

Granular cell carcinoma. Intense granular staining is found in all cells (arrowheads). Original magnification ×40.

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 acid-base 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 (70–80%) 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.

Footnotes

Acknowledgements

Supported by grants from the Ministerio de Ciencia y Tecnología (BMC2003-03297), the Comunidad de Madrid (08.5/0066.1/2001), Fondo de Investigación Sanitaria (01/0085-01 and -02), and Fundación Eugenio Rodriguez Pascual.

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