Crossreaction with an Anti-Bax Antibody Reveals Novel Multi-endocrine Cellular Antigen
Department of Anatomy and Cell Science, Kansai Medical University, Moriguchi-City, Osaka (KU,JW,YT,YK,HY) and Department of Anatomy, Shiga University of Medical Science, Otsu-City, Shiga (KK), Japan
Correspondence to: Hisao Yamada, MD, PhD, Dept. of Anatomy and Cell Science, Kansai Medical University, Fumizono-cho, Moriguchi-City, Osaka 570-8506, Japan. E-mail: yamada{at}takii.kmu.ac.jp
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Summary |
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(J Histochem Cytochem 52:805812, 2004)
Key Words: Bax cross-immunoreactivity immunohistochemistry hypothalamus pituitary gland pancreatic islet ACTH -MSH glucagon pro-opiomelanocortin (POMC)
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Introduction |
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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).
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Materials and Methods |
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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 ultramicrotome, 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 4361 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 avidinbiotinperoxidase 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 x 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.
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Results |
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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 hypothalamoposterior 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 -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).
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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.
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Discussion |
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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 hypothalamoposterior pituitary system, in all ACTH (-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 -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
-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
-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 hypothalamoposterior 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 4361 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.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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