ARTICLE |
Correspondence to: Jacques Baudier, Departement Reponse et Dynamique Cellulaires, TS EMI 0104, DRDC, CEA Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France. E-mail: jbaudier@cea.fr
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Summary |
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Here we report a detailed analysis of the expression and localization of the giant protein AHNAK in adult mouse tissues. We show that AHNAK is widely expressed in muscle cells, including cardiomyocytes, smooth muscle cells, skeletal muscle, myoepithelium, and myofibroblasts. AHNAK is also specifically expressed in epithelial cells of most lining epithelium, but is absent in epithelium with more specialized secretory or absorptive functions. In all adult tissues, the main localization of AHNAK is at the plasma membrane. A role for AHNAK in the specific organization and the structural support of the plasma membrane common to muscle and lining epithelium is discussed.
(J Histochem Cytochem 51:339348, 2003)
Key Words: muscle, AHNAK, epithelium, tissue localization
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Introduction |
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THE GIANT PROTEIN AHNAK has been identified by in the presence of arachidonic acid (
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Materials and Methods |
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Antibodies and Controls
Rabbit polyclonal antibodies were made against AHNAK using two different epitopes. The first, KIS (CKISMPDVDL HLKGPK), was chosen according to the strategy of
Immunoblotting
In adult mouse, specimens from kidney, heart, thymus, esophagus, pancreas, liver, and lung were removed. These specimens were cut into small pieces and quickly frozen with liquid nitrogen until used. Specimens were homogenized with a glass homogenizer and were then heated at 90C for 3 min in denaturation buffer composed of 2% SDS, 25 mM Tris-HCl, pH 6.8, 10% glycerol, 0.1% bromophenol blue, 1m M EDTA. Proteins were electrophoretically resolved by one-dimensional SDS-PAGE (6% polyacrylamide), the gel was then equilibrated in transfer buffer containing 0.03% SDS and proteins were transferred onto nitrocellulose membrane. The membrane was first incubated with CQL-AHNAK antibody (1:100 dilution) in TBS containing 0.3% Tween-20 (TTBS) at 4C for 16 hr. After washing, the membrane was incubated with a 1:10,000 dilution of horseradish peroxidase secondary antibody and the immune complex was revealed by chemiluminescence. Nitrocellulose membrane was then stripped in TTBS with 1% SDS and 0.1% sodium azide, and then reprobed with the KIS-AHNAK antibody (1:1000 dilution).
Immunochemistry
Tissues were screened for AHNAK localization using a tissue array produced by ResGen Co. (Invitrogen; Carlsbad, CA). We used paraffin-embedded tissue spots representing several mouse organs: adrenal, brain, heart, large intestine, small intestine, renal cortex, renal medulla, liver, lung, lymph node, skeletal muscle, pancreas, salivary gland, skin, spleen, stomach, thymus, prostate, testis, ovary, uterus, breast, and esophagus. Paraffin was removed and immunohistochemistry was then performed using the DAKO ABC horseradish peroxidase system (DAKO; Carpinteria, CA). After endogenous peroxidase inactivation with H2O2 3% and sodium azide 0.05%, followed by extensive washing, tissues were blocked in 5% normal goat serum in TBS for 30 min and incubated with the KIS-AHNAK antibody at 1:100 dilution in TBS plus 2% normal goat serum overnight at 4C. After rinsing with TBS, tissues were incubated with a biotinylated goat anti-rabbit Ig (DAKO) diluted 1:100 in TBS plus 5% normal goat serum for 30 min at room temperature. After extensive washing with TBS, the slide was incubated with the horseradish peroxidase complex according to the manufacturer's recommendations. The chromogen used was AEC+ (DAKO). Slides were counterstained with hematoxylin. For indirect immunofluorescence localization of AHNAK, mouse testis, brain, and liver were frozen in isopentane at -80C and cut in 10-µm sections. Slides were fixed with 4% paraformaldehyde for 30 min and washed abundantly before permeabilization for 5 min with 0.5% Triton X-100 in TBS. After extensive washing and blocking with goat serum, primary antibodies were incubated overnight at 4C at the following dilutions: KIS-AHNAK 1:100; PECAM 1:5. Slides were then incubated with secondary antibody for 1 hr and nuclear countstaining was performed with Hoechst 33258 (1 µg/ml). Double immunofluorescence analysis was performed using confocal microscopy (Leica TCS-SP2) or fluorescence microscopy (Zeiss Axiovert 200M). Images were directly captured, saved, and transferred to Adobe Photoshop 5.5.
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Results |
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Immunoblotting Analysis and Tissue Distribution of AHNAK
Extracts from various mouse tissues were analyzed for AHNAK expression by immunoblotting using affinity-purified antibodies raised against the CQL and the KIS peptides (Fig 1). To normalize for total protein content of the tissue preparations, the extracts were also analyzed for -tubulin content (Fig 1A). Both the CQL and the KIS antibody reacted strongly with a major protein band that migrated with the expected molecular weight for AHNAK, confirming the specificity of our antibodies (Fig 1B). A high level of expression of AHNAK was observed in heart, lung and esophagus, whereas kidney, pancreas, and liver expressed only moderate levels of AHNAK. AHNAK immunoreactivity was absent in the thymus. CQL and KIS antibodies also reacted with lower molecular weight protein bands. These protein bands are present only in tissue extracts that express the full-length AHNAK protein and may therefore represent proteolytic products of AHNAK. In support of that assumption, we found that AHNAK is highly sensitive to metal-dependent proteases. When tissues are lysed in the absence of the metal chelators EDTA and EGTA, the full-length AHNAK immunoreactivity decreases, with a concomitant increase in the lower molecular weight protein bands recognized by both the AHNAK KIS and CQL antibodies (data not shown). A band of approximately 100 kD was also detected with the CQL antibody but not with the KIS antibody. This band might correspond to a C-terminal AHNAK proteolytic fragment. The affinity-purified KIS antibody was next used for immunoperoxidase and indirect immunofluorescence analysis of AHNAK immunoreactivity in mouse tissues (Fig 2 Fig 3 Fig 4 Fig 5).
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AHNAK Localization in Muscle Cells
Immunohistochemical analysis of AHNAK immunoreactivity in different tissues revealed a specific localization of AHNAK in muscle cells (Fig 2). As previously reported by
AHNAK Localization in Lining Epithelium
In the stratified epithelium of the skin, AHNAK was expressed at the plasma membrane of keratinocytes of the stratum basale and the stratum spinosum (Fig 3A). AHNAK staining was not observed in the cornified layer. AHNAK is also expressed in cells of papillary dermis and in some cells of the hair follicle (Fig 3A).
In the upper part of the digestive tract, i.e., the esophagus, AHNAK was found in the stratified squamous epithelium, which in rodents may be keratinized (Fig 3B). Here again, AHNAK immunoreactivity was concentrated mainly at the plasma membrane. As shown in Fig 3B, AHNAK was also present in the muscle cells of the striated muscularis layer. In kidney, AHNAK immunochemistry showed that AHNAK was localized in a special type of epithelium of the urinary system that lines the collecting ducts, ducts of Bellini, and the pelvicalyceal system (Fig 3D; and data not shown). This transitional epithelium, called urothelium, is present along the conducting passages of the urinary system and protects the organism from acid and hypertonic urine. In liver, AHNAK immunoreactivity was confined to the portal tract. Fig 3E focuses on a typical portal tract containing three main structures. The largest is a terminal branch of the hepatic portal vein (PV), which has a thin wall lined by endothelial cells labeled with PECAM antibody. These endothelial cells were AHNAK-positive. Smaller-diameter vessels are arterioles, which show lower AHNAK immunostaining. Finally, the bile collecting duct, lined by simple columnar epithelium, was strongly immunostained with AHNAK antibody. In these lining epithelial cells, AHNAK was concentrated at the plasma membrane. There was no AHNAK immunostaining of the liver hepatocytes. Immunohistochemistry with the lung (Fig 3E) confirmed the Western blotting analysis, showing that a high level of AHNAK protein is present in lung (Fig 1, Lane 3). AHNAK was highly expressed in parenchymal cells (epithelium of alveoli) and in the smooth muscle layer surrounding the bronchiolar epithelium. Faint AHNAK immunoreactivity was also present in pseudostratified bronchial epithelium. The labeled epithelial cells were mainly ciliated cells characterized by large nuclei. Their soma was weakly immunostained but concentrated immunoreaction deposit was observed at the apical plasma membrane and in contact with the basement membrane (white arrow).
We also found AHNAK immunoreactivity associated with the plasma membrane of several other lining epithelia, such as the stratified epithelium of the tunica albuginea of the testis (data not shown), and also in the epithelial cells lining the ventricles of the central nervous system (Fig 4). In the adult mouse brain, AHNAK immunoreactivity was abundant in epithelial cells that bordered all the ventricles, including the lateral ventricles (not shown), the third ventricle (Fig 4A), and the fourth ventricle (Fig 4B). There was no apparent AHNAK immunostaining of neuronal and glial cell soma. Brain capillary endothelial cells were also immunostained (see Fig 4A).
In contrast to muscle cells, not all epithelial cells synthesize AHNAK. As shown in Fig 5A, the different secretory epithelial cells that constitute the stomach mucosa, including mucus-secreting cells, acid-secreting cells, and pepsin-secreting cells, were not AHNAK-immunoreactive. AHNAK staining appeared as a thin strand between the gastric gland, which corresponds to the muscularis mucosae that extends from the base of a gastric gland, to the lumen, whose contraction expels gastric secretion into the stomach lumen. The absence of AHNAK in secretory epithelium was confirmed in the salivary gland (Fig 5B), the mammary gland (Fig 5C), and the prostate gland (Fig 5D). In the salivary gland, the epithelium of serous acini, with nuclei flattened against the basement membrane, and the epithelium of mucous acini were not AHNAK-immunoreactive. The thin AHNAK immunoreactivity surrounding serous acini corresponds to the processes of contractile myoepithelial cells. In mammary glands, myoepithelial cells surrounding the gland and endothelial cells of capillaries were also labeled by the AHNAK antibody (Fig 5C). The prostate glandular epithelium was not positive for AHNAK, whereas the supporting tissue, which contains numerous smooth muscle cells and capillaries, was labeled by the AHNAK antibody (Fig 5D). Fig 5E shows the immunolabeling of AHNAK in the pancreas, a large gland composed of glandular epithelium with exocrine and endocrine functions. The endocrine tissue formed by the islets of Langerhans was not stained, nor were the glandular acini, which constitute the exocrine part of the pancreas. Only the endothelial cells of the capillaries were immunolabeled. AHNAK immunostaining was also not observed in epithelium with absorptive functions, such as the epithelium of the small intestine (data not shown; see also Fig 2D) and the absorptive epithelium lining the colon, which also has protective functions (Fig 5F). The mucosa of the colon, formed of straight tubular glands consisting of cells specialized for water absorption and of mucus-secreting globlet cells, was devoid of AHNAK immunoreactivity. However, significant cytoplasmic immunoreactivity was present in the cells of the luminal portion of the epithelium. These cells, which are derived from precursor cells present in the basal portion of the epithelium, are more mature and are subjected to the stretching forces of the luminal content. The muscularis mucosae, which extends into the lamina propria between the folds of the gland, also expressed AHNAK.
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Discussion |
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Table 1 summarizes the tissue distribution and the cellular localization of AHNAK in mouse tissues. The protein AHNAK is a hallmark of muscular and muscle-like cells with contractile properties organized in contractile tissue or scattered in the tissue as fibromyocytes or myoepithelial cells. AHNAK is also specifically expressed in epithelial cells of the skin, the esophagus, the tunica albugina of the testis, the bile ducts, and the brain ventricles. In contrast to muscle cells, not all epithelial cells express AHNAK. We did not find significant AHNAK immunoreactivity in the glandular epithelium of the mammary gland, the salivary gland, the stomach, the prostate, or the exocrine and endocrine pancreas, all of which have secretory functions. We were also unable to detect AHNAK in the absorptive epithelium of the proximal part of the nephron and of the intestine. In addition to muscle cells and lining epithelial cells, AHNAK is also present in endothelial cells of several capillary and blood vessels (see Fig 2F, Fig 3D, Fig 4A, and Fig 5C5E). However, in fenestrated endothelium of the kidney glomeruli (Fig 3C), the hepatic sinusoid (data not shown), and the continuous capillaries of lung (data not shown), AHNAK immunoreactivity was below the detection limit. This suggests that the level of AHNAK expression in endothelial cells might depend on the blood vessel properties.
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In adult tissue, the main subcellular localization of AHNAK is at the plasma membrane. Cytoplasmic staining was also observed in fibroblast or certain epithelial cells, such as those in the colon (Fig 5F). Exclusive membrane location of AHNAK has also been recently reported in rat cardiomyocytes by
The predominant cellular expression of AHNAK in muscles and in lining epithelium that we report in this study might help to elucidate the membrane function of this giant protein. First, it allows restriction of the search for AHNAK function to essentially two specialized cell types. Moreover, several structural and functional features are shared between muscle and lining epithelium. These tissues are subjected to stretching force and have elastic properties. For example, the epithelium of the esophagus is subjected to the stretching force of the bolus, and muscle cells to stretching forces during the contractionrelaxation cycles. This implies that common cellular organizations are involved in both tissues to respond and control the stretching forces. The first is the cellcell interactions necessary for maintaining tissue integrity. Several molecular structures are known to participate in cellcell interactions, such as desmosomes, tight junctions, and adherens junctions. Although initially AHNAK was co-purified with the desmosomal component during cell fractionation of keratinocytes ( in the presence of arachidonic acid (
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Acknowledgments |
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Supported in part by a grant from Association pour la Recherche contre le Cancer (Christian Delphin) and a Ligue Nationale contre le Cancer fellowship (Benoit Gentil).
We thank Dr Peoc'h for valuable advice and Dr La Marre Jonathan for critical reading of the manuscript.
Received for publication July 25, 2002; accepted October 9, 2002.
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