Crossreaction with an Anti-Bax Antibody Reveals Novel Multi-endocrine Cellular Antigen

Abstract

We found a novel protein that has crossreactivity with a polyclonal anti-Bax antibody (SCBAX antibody). The protein was localized exclusively in the endocrine cells of hypothalamus, pituitary gland, and pancreatic islets. Immunohistochemical (IHC) double labeling revealed that the cells showing crossreactivity with this antibody corresponded precisely to oxytocin neurons and ACTH, α-MSH, and glucagon cells in rat and gerbil. By immunoelectron microscopy, the protein was localized predominantly in and just around the secretory granules in the cytoplasm but not in the mitochondria. Double-labeling IHC with the anti-Bax SCBAX antibody and two anti-Bax monoclonal antibodies (MAbs) showed that cells stained with the anti-Bax SCBAX antibody were not stained with anti-Bax MAbs except for very few cells (probably apoptotic cells). Western blotting analysis revealed that the molecular mass of the protein was ∼55 kD, which differs from that of Bax protein (21 kD). These findings indicate that the anti-Bax SCBAX antibody recognizes not only proapoptotic Bax protein (a 21-kD mitochondrial protein) but also an unknown substance present in one endocrine cell group in each endocrine organ. Therefore, the protein is designated as multi-endocrine cellular antigen (MECA). MECA is probably a 55-kD protein secreted from the particular differentiated cell groups of endocrine tissues. (J Histochem Cytochem 52:805–812, 2004)

Keywords

Bax cross-immunoreactivity immunohistochemistry hypothalamus pituitary gland pancreatic islet ACTH α-MSH glucagon pro-opiomelanocortin (POMC)

Bcl-2-associated X protein (Bax) is one of the proapoptotic members of the Bcl-2 family, which participates in active cell turnover by counteracting the protective effects of Bcl-2 against apoptotic cell death (Oltvai et al. 1993). Immunohistochemical (IHC) studies have revealed the distribution of Bax-like immunoreactivity in several mammalian organs, such as hematolymphoid tissues (Ohta et al. 1995), the cardiovascular system, and reproductive tissues (Krajewski et al. 1994).

We had previously examined distribution of the immunoreactivity to anti-Bax SCBAX antibody and had detected Bax-like reactivity in a large number of magnocellular neurons in the rat hypothalamo-posterior pituitary system (Matsuda et al. 2001). We also showed the possibility that a Bax-like reactive substance is secreted into the circulatory system (Matsuda et al. 2001). Using the same antibody, Sugimoto et al. (1996) reported strong Bax-like immunoreactivity in the neurons of the primary sensory cortex and the hypothalamic neuroendocrine nucleus. These neurons, including hypothalamic magnocellular neurons, do not undergo active cell turnover under non-pathological conditions (Sturrock 1979; Mesulam et al. 1987; Pugnaloni et al. 1998). These findings suggest that the anti-Bax SCBAX antibody recognizes not only the pro-apoptotic Bax protein (mitochondrial protein, ca. 21 kD) but also another antigen. It is therefore interesting to determine the histochemical and biochemical characteristics of this unknown antigen that crossreacts with the SCBAX antibody.

In the present study we used IHC and Western blotting analysis to show that the SCBAX-crossreacting antigen is distinct from the pro-apoptotic Bax protein, to examine its distribution, and to characterize the endocrine cell types in which it is expressed. We designated this novel protein as multi-endocrine cellular antigen (MECA).

Materials and Methods

Experimental Animals

All animals (male Wistar rats weighing 300–350 g and male Mongolian gerbils weighing 65–80 g; Nippon SLC, Shizuoka, Japan) were maintained at the Institute for Experimental Animals of the University under the Guidelines for Animal Experimentation at Kansai Medical University, based on the guidance of the Japanese Association for Laboratory Animal Science.

Experiments were performed on (a) 10 rats and 20 gerbils for IHC and double-labeling IHC at the light microscopic level, (b) 15 rats for Western blotting analysis, and (c) 10 gerbils for immunoelectron microscopy. Before each experimental procedure, all animals were deeply anesthetized with sodium pentobarbital (70 mg/kg body weight IP).

Tissue Preparation for Histochemistry

Each animal was perfused transcardially with 0.1 M PBS under deep anesthesia, followed by a fixative containing 4% formaldehyde and 0.2% picric acid in 0.1 M phosphate buffer at pH 7.4. The brain, pituitary, thyroid and parathyroid glands, pancreas, adrenal glands, lung, heart, liver, gastrointestinal tracts, kidney, and skeletal muscle were quickly dissected, then immersed in the same fixative for 12 hr. After fixation these organs were cryoprotected in 20% sucrose solution, frozen with CO₂ gas, and cut with a cryostat into 18-μm sections. Immunostaining was performed by the free-floating ABC method (Yamada et al. 1992).

For immunoelectron microscopy, the pituitary glands were cut with a microslicer into 500-μm sections. These sections were dehydrated, embedded in LR Gold resin (London Resin Company; London, UK), and then polymerized with a UV lamp. Ultrathin sections (90 nm) were cut with an ultra-microtome, mounted on nickel grids, and subjected to immunocytochemical staining.

Immunohistochemistry

Anti-Bax SCBAX antibody (sc-526; Santa Cruz Biotechnology, Santa Cruz, CA) is an affinity-purified polyclonal antibody raised against a peptide corresponding to amino acids 43–61 of mouse Bax protein. The specificity for epitope peptide was confirmed by an immunoabsorption test in which the immunogen peptide (sc-526P; Santa Cruz Biotechnology) was added to the antibody before IHC. The peptide caused dose-dependent attenuation of IHC staining.

Frozen sections were incubated with the anti-Bax SCBAX antibody (diluted 1:1000) for 48 hr at 4C, biotin-labeled anti-rabbit antibody for 3 hr at room temperature (RT), and avidin-biotin-peroxidase complex solution for 1.5 hr at RT. Finally, sections were exposed to 3,3′-diaminobenzidine tetrahydrochloride (DAB) solution and Bax immunoreactivity was visualized as brown.

Double-labeling Immunohistochemistry

In the first staining procedure, the sections were incubated with the anti-Bax SCBAX antibody (rabbit, 1:1000) for 48 hr at 4C, followed by incubation with Cy2-conjugated anti-rabbit antibody (goat; Jackson ImmunoResearch, West Grove, PA; 1:100) for 3 hr at RT. In the second staining procedure, the sections were incubated with each anti-Bax MAb (anti-Bax YTH-6A7 antibody, mouse; Trevigen, Gaithersburg, MD; 1:500, and anti-Bax 4F11 antibody, mouse; Immunotech, Marseilles, France; 1:500) or anti-hormone antibodies (described below) for 24 hr at 4C, followed by incubation with Cy5-conjugated antibodies (described below) for 3 hr at RT. They were examined with a confocal laser scanning microscope (LSM510-Ver. 2.8; Carl Zeiss, Oberkochen, Germany).

The anti-hormone antibodies used were anti-oxytocin (mouse; Chemicon International, Temecula, CA; 1:2000), anti-Arg-vasopressin (rabbit; Chemicon; 1:7000), anti-ACTH (mouse; Biogenesis, Poole, UK; 1:1000), anti-GH (rabbit; Chemicon; 1:3000), anti-TSH (rabbit; Biogenesis; 1:7000), anti-prolactin (rabbit; Biogenesis; 1:2000), anti-FSH and anti-LH (rabbit; Chemicon; 1:5000), anti-glucagon (goat; Santa Cruz Biotechnology; 1:2000), anti-somatostatin (rabbit; Affinity Research Products, Exeter, UK; 1:5000), and anti-insulin (guinea pig; Novo Industri, Bagsvaerd, Denmark; 1:2000). Secondary antibodies used were goat anti-mouse, goat anti-rabbit, and donkey anti-guinea pig conjugated with Cy5 (Jackson ImmunoResearch; 1:100).

Immunoelectron Microscopy

The sections on nickel grids were incubated in mixtures of SCBAX antibody (rabbit, 1:500) with anti-oxytocin (mouse, 1:2000) or anti-ACTH (mouse, 1:1000) antibody for 12 hr at 4C. The sections were then incubated in mixtures of 15-nm gold-labeled anti-rabbit (goat; ICN Biomedicals, Aurora, OH; 1:200) and 5-nm gold-labeled anti-mouse (goat; ICN Biomedicals; 1:200) antibodies for 3 hr at RT. The sections were stained with uranyl acetate (Merck; Darmstadt, Germany) and examined with an electron microscope (H-7100; Hitachi, Tokyo, Japan).

Western Blotting Analysis

The animals were perfused transcardially with 0.1 M PBS. The hypothalami, including paraventricular nucleus (PVN), pituitary glands, and pancreas were quickly dissected, homogenized in 0.01 M Tris-HCl buffer at pH 7.4 containing 0.2% Triton X-100, 5 mM NaCl, 5 mM MgCl2, and protease inhibitor cocktail (Complete Mini; Roche Molecular Biochemicals, Mannheim, Germany) at 4C. The suspensions were centrifuged at 3000 × g for 10 min and the supernatants were collected. The protein content in the supernatant was measured by the Lowry method. The protein samples (50 μg) were separated by SDS-PAGE (7.5% acrylamide) and transferred to a PVDF transfer membrane (Immobilon-P; Millipore, Bedford, MA). The membrane was soaked in SCBAX antibody (1:1000) for 12 hr at RT and in HRP-conjugated goat anti-rabbit antibody (Zymed Labs, San Francisco, CA; 1:1000) for 3 hr at RT. The membrane was exposed to the Konica Immunostaining HRP-1000 solution (Konica; Tokyo, Japan) and Bax immunoreactivity was visualized as blue.

Figure 1

Photomicrographs showing cells stained with the anti-Bax SCBAX antibody in the endocrine organs of rats (left; A,C,E,G) and gerbils (right; B,D,F,H). Many immunopositive neuronal cell bodies and fibers were present in the PVN (caudal area A; rostral area B) and SON (rostral area C; caudal area D). A large number of immunoreactive axon terminals were in the pituitary posterior lobe, and immunopositive cells were also observed in the pituitary anterior and intermediate lobes (E,F). The immunopositive cells were distributed in the peripheral portion of the pancreatic islet (G,H). All photographs are at the same magnification. Bars = 100 μm.

Results

Distribution of Immunoreactivity with the Anti-Bax SCBAX Antibody

In rats and gerbils, we evaluated the precise distribution of immunoreactive cells using the anti-Bax SCBAX antibody in endocrine tissues and several other organs. Intense immunoreactivity was observed as dotlike granules in the cytoplasm of endocrine cells. The immunopositive cells were found only in selected endocrine tissues: the hypothalamo–posterior pituitary system, pituitary glands, and pancreatic islets (Figure 1). No immunoreactivity was detected in the other organs tested: pineal body, thyroid and parathyroid glands, adrenal glands, gastrointestinal tract, lung, heart, liver, kidney, and skeletal muscle. These histological features in rats (Figures 1A, 1C, 1E, and 1G) and gerbils (Figures 1B, 1D, 1F, and 1H) were the same. In the hypothalamo–posterior pituitary system, many immunopositive neurons were present in the paraventricular nucleus (PVN), including most magnocellular neurons and some parvocellular neurons (Figures 1A and 1B), and the supraoptic nucleus (SON) of the hypothalamus (Figures 1C and 1D). In these neurons the immunoreactivity was observed in the perikarya and nerve fibers with Herring's bodies. Immunopositive nerve fibers were also observed in the internal layer of the median eminence (ME). Many immunopositive axon terminals were located in the posterior pituitary (Figures 1E and 1F). Immunopositive cells were also observed in the anterior and intermediate lobes of pituitary glands (Figures 1E and 1F). The relative staining intensity of the anterior lobe was high, whereas that of the intermediate lobe was weak to moderate. In the pancreatic islets, the immunopositive cells were located in the peripheral part of each pancreatic islet (Figures 1G and 1H).

Histochemical and Biochemical Differences from Authentic Bax Protein

To test whether the intense immunoreactivity with the anti-Bax SCBAX antibody in endocrine tissues was caused by a protein distinct from Bax, the distribution of SCBAX immunoreactivity was compared with that of Bax-specific MAbs using a double-labeling technique (Figure 2). In the anterior pituitary, a large number of cells were densely labeled with the SCBAX polyclonal antibody (Figure 2A), while only a few cells were stained with the anti-Bax YTH-6A7 MAb (Figure 2B). In the merged image (Figure 2C), a few cells positive for the anti-Bax YTH-6A7 antibody also reacted with the SCBAX antibody, whereas many other cells positive with the SCBAX antibody were not stained with anti-Bax YTH-6A7. Double labeling using the SCBAX antibody with the anti-Bax 4F11 MAb yielded similar results (data not shown).

Figure 2

Double-labeling immuno-fluorescence images showing cells stained with the anti-Bax SCBAX antibody (green) and anti-Bax YTH-6A7 antibody (red) in the pituitary anterior lobe of the gerbil. A large number of cells were stained with the anti-Bax SCBAX antibody (A), whereas a few cells were labeled with anti-Bax YTH-6A7 monoclonal antibody (B). Only two cells were double labeled (C). Western blotting analysis with the anti-Bax SCBAX antibody in the rat endocrine tissues (D). Tissues used were the hypothalamus (Lane 1), pituitary (Lane 2), and pancreas (Lane 3). Positions of molecular weight markers are indicated in kilodaltons.

Western blotting analysis with the anti-Bax SCBAX antibody showed a major band of ∼55 kD (Figure 2D) from the hypothalamus (Figure 2D, Lane 1), pituitary gland (Figure 2D, Lane 2), and pancreas (Figure 2D, Lane 3). A minor band of ∼21 kD, corresponding to authentic Bax protein, and some other bands were faintly detected (Figure 2D, Lanes 1–3), while only a major band of ∼21 kD was labeled with the anti-Bax YTH-6A7 and 4F11 MAbs (data not shown).

Figure 3

(A) Double-labeling immunofluorescence images showing cells stained with the anti-Bax SCBAX antibody (green, left column) and anti-hormone antibodies (red, middle column) in gerbil endocrine organs. The double-labeled cells are seen as yellow in the merged images (right column). The cells containing immunoreactivity to the SCBAX antibody (SCBAX) were identical to the oxytocin neurons in the PVN (top panels), to ACTH cells in the pituitary anterior lobe (middle panels), and to glucagon cells in the pancreatic islet (bottom panels). (B) Merged double-labeling images of gerbil tissue stained with anti-Bax SCBAX antibody (green) and various anti-hormone antibodies (red). Shown are the Arg-vasopressin neurons in the PVN, POMC neurons in the arcuate nucleus of the hypothalamus, the prolactin-, GH-, FSH/LH-, and TSH-containing cells in the pituitary anterior lobe, and the insulin- and somatostatin-containing cells in the pancreatic islets. Bars = 50 μm.

Identification of Endocrine Cell Type

Double-labeling IHC with the anti-Bax SCBAX antibody and anti-hormone antibodies showed that the immunoreactivity with the SCBAX antibody was exclusively localized in one type of endocrine cell group per endocrine organ (Figures 3A and 3B). In the hypothalamo-posterior pituitary system, the neurons showing immunoreactivity to the SCBAX antibody corresponded exactly to the oxytocin-containing neurons in the hypothalamus, including the PVN (Figure 3A), but did not correspond to the Arg-vasopressin neurons (Figure 3B). In the pro-opiomelanocortin (POMC)-containing arcuate hypothalamic nucleus (Figure 3B), no neurons showed immunoreactivity to the SCBAX antibody. In the pituitary glands, the endocrine cells labeled with the SCBAX antibody were identical to the ACTH cells of the anterior lobe (Figure 3A) and the α-MSH cells of the intermediate lobe. The prolactin, GH, FSH/LH, and TSH cells showed no immunoreactivity to the SCBAX antibody (Figure 3B). In the pancreatic islets, the set of endocrine cells stained with this antibody showed 100% correspondence with the glucagon cell group (Figure 3A) but not with the insulin or somatostatin cell groups (Figure 3B).

In the POMC-expressing lineages, the staining intensity with the SCBAX antibody was high in the ACTH cells (Figure 3A), weak to moderate in the α-MSH cells, and negative in the POMC neurons of the arcuate hypothalamic nucleus (Figure 3B).

Subcellular Distribution of the Protein Labeled with the Anti-Bax SCBAX Antibody

In the anterior pituitary, the SCBAX immunoreactivity was predominantly localized in and just around the secretory granules of the ACTH cells but not on the mitochondria (Figure 4). Secretory granules immunopositive for the SCBAX antibody were also labeled with the anti-ACTH antibody, but the staining intensity in each secretory granule labeled with the SCBAX antibody did not correlate with the intensity stained with the ACTH antibody.

In the posterior pituitary stained with the SCBAX antibody, the immunoreactive and non-immunoreactive axon terminals were intermingled. In the hypothalamic endocrine neurons stained with the anti-oxytocin antibody, the SCBAX immunoreactivity was also predominantly localized in and just around the secretory vesicles. Some axon terminals containing the SCBAX-immunopositive secretory vesicles adjoined the pericapillary space.

Figure 4

Double-labeling immuno-electron microscopy shows the immunoreactivity with the anti-Bax SCBAX antibody (large gold particles) and anti-ACTH antibody (small gold particles) in the pituitary anterior lobe of the gerbil. Immunolabeling was exclusively in and just around the secretory granules but not on the mitochondrion (arrowhead) in the ACTH cells. N, nucleus. Bar = 1 μm.

Discussion

The Bax protein has been identified as a regulator of apoptotic cell death (Oltvai et al. 1993). By IHC, many researchers reported the widespread distribution of Bax protein in several organs (Krajewski et al. 1994; Ohta et al. 1995). Using the anti-Bax SCBAX antibody, two different groups reported that Bax-like immunoreactivity was present in the neurons and that Bax-like protein was probably secreted from hypothalamic neurons (Sugimoto et al. 1996; Matsuda et al. 2001). Our present study further showed that intense immunoreactivity with the anti-Bax SCBAX antibody was present not only in the hypothalamo-posterior pituitary system but also in the pituitary glands and pancreatic islets. Endocrine cells, especially the hypothalamic magnocellular neurons, do not undergo active cell turnover under non-pathological conditions (Sturrock 1979; Mesulam et al. 1987; Pugnaloni et al. 1998). These findings support the hypothesis that the anti-Bax SCBAX antibody recognizes not only proapoptotic Bax protein but also another antigen. In this study we used several experimental procedures to analyze the phenomenon of SCBAX immunoreactivity in many cells in specific endocrine organs and obtained new data indicating that it resulted from cross-immunoreactivity, for the following reasons. First, the cells labeled with the anti-Bax SCBAX antibody were not stained by anti-Bax MAb, except for a very few cells (probably apoptotic cells). Western blotting analysis revealed that the anti-Bax SCBAX antibody recognized a protein of ∼55 kD, whereas authentic Bax protein is 21 kD. Finally, by immunoelectron microscopy, the protein densely labeled by the anti-Bax SCBAX antibody was localized in and just around the secretory granules but not on the mitochondria, although the cytotoxic function of pro-apoptotic Bax protein depends on its redistribution from cytosol to the mitochondrial membrane (Marzo et al. 1998; Murphy et al. 1999). These findings show that the Bax-like protein labeled with the anti-Bax SCBAX antibody appears to be a novel protein of ∼55 kD that differs from the Bax protein. Therefore, we designated this novel protein as multi-endocrine cellular antigen (MECA) because it appeared exclusively in several endocrine organs.

Subsequently, to investigate roles for MECA, we have identified the cell types containing MECA. IHC double labeling with the anti-Bax SCBAX antibody and anti-hormone antibodies showed that MECA was exclusively localized to all of the oxytocin neurons in the hypothalamo–posterior pituitary system, in all ACTH (α-MSH) cells in the pituitary glands, and in all glucagon cells in the pancreatic islets. These findings suggest that MECA is present in one endocrine cell group in each endocrine organ.

The anti-Bax SCBAX antibody has commonly been used in various investigations of pro-apoptotic Bax protein. Ahlbom et al. (1998) have shown the presence of many cells immunopositive with the SCBAX antibody in the anterior pituitary of rat. They believed that the immunopositive cells were prolactin-containing lactotrophs because the immunostaining was altered during lactation. However, they did not confirm the cell type using double labeling.

POMC-containing cells have been located in the pituitary gland and the brain. Double-labeling IHC revealed that the staining intensity of MECA varied in the different POMC-expressing lineages, i.e., high levels in the ACTH cells of pituitary anterior lobe, weak to moderate levels in the α-MSH cells of pituitary intermediate lobe, and negative in the POMC neurons in the hypothalamus. These findings indicated that MECA is not the same as ACTH itself, a precursor, or a related peptide. Recent studies demonstrate that cell-specific transcription of pituitary hormone-coding genes relies on a combination of cell-restricted and tissue (pituitary)-restricted T box transcription factors (Kioussi et al. 1999; Sheng and Westphal 1999; Scully and Rosenfeld 2002). A pituitary cell-restricted T box factor, Tpit, activates POMC transcription in cooperation with Pitx homeo-proteins (Lamolet et al. 2001). In developing and adult mammals, Tpit is present only in the two pituitary POMC-expressing lineages, i.e., in all of the ACTH cells in the anterior lobe and in the α-MSH cells in the intermediate lobe, and apparently in no other tissues, including the POMC neurons in the brain. Moreover, in the adult pituitary, IHC has shown high levels of Tpit expression in the ACTH cells and moderate levels in the α-MSH cells. In the POMC-expressing lineages, the pattern of MECA expression is very similar to that of Tpit. These findings support the hypothesis that MECA might be induced by the cell type-specific T Box factor (i.e., Tpit) and might play important roles for terminal cell-type differentiation in particular types of endocrine cells. At a minimum, MECA can be used as a cell-type differentiation marker in the hypothalamo–posterior pituitary system, pituitary glands, and pancreatic islets.

In the pituitary, MECA was predominantly localized in and just around the secretory granules but not on the mitochondria in the ACTH cells and oxytocin neurons. Moreover, in the posterior pituitary some axon terminals containing Bax-like immunoreactivity adjoined the pericapillary space (Matsuda et al. 2001). Our previous study using the EIA method (Matsuda et al. 2001) also revealed that the average concentration of Bax-like immunoreactivity in the serum was 39.0 ± 9.6 pmol/ml. In animals with axon flow pharmacologically disturbed by colchicine, the densities of immunoreactivity were increased in the PVN and the serum levels were decreased to 29.5 ± 5.3 pmol/ml compared with non-treated animals. These results strongly suggested that MECA is a secretory protein.

In conclusion, we found a novel 55-kD protein that showed crossreactivity with the anti-Bax SCBAX antibody. The protein was exclusively localized in the endocrine cells and was designated as multi-endocrine cellular antigen (MECA). MECA was present in a single endocrine cell group in each endocrine gland and may be closely correlated with cell-type differentiation in particular types of endocrine cells. Moreover, MECA was detected in and just around the secretory granules and might possibly be a secretory protein secreted into the systemic circulation.

Several Bax family proteins were found with the database analysis (BLAST and FASTA) for partial overlapping sequence for 43–61 amino acids of mouse Bax protein, which is the immunogen for the anti-Bax SCBAX antibody. To determine the precise chemical structure of MECA, we are now trying to isolate MECA mRNA by immunoscreening. After complete identification and chemical determination of MECA, we shall confirm the MECA-containing cells using the antibody to the authentic MECA.

Footnotes

Acknowledgements

Supported by a Grant-in-Aid for Young Scientists (B), no. 14770010, from the Japan Society for Promotion of Science (to YT), and by a grant, Research Grant B, from Kansai Medical University (to HY).

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