ARTICLE |
Correspondence to:
John Y.H. Chan, Inst. of Radiological Sciences, National Yang Ming University, Shi-Pai, Taipei 112, Taiwan ROC. E-mail: chanyhjohn@hotmail.com
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Summary |
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Genes that modulate the action of hormones and cytokines play a critical role in stress response, survival, and in growth and differentiation of cells. Many of these biological response modifiers are responsible for various pathological conditions, including inflammation, infection, cachexia, aging, genetic disorders, and cancer. We have previously identified a new gene, BRE, that is responsive to DNA damage and retinoic acid. Using multiple-tissue dot-blotting and Northern blotting, BRE was recently found to be strongly expressed in adrenal cortex and medulla, in testis, and in pancreas, whereas low expression was found in the thyroid, thymus, small intestine and stomach. In situ hybridization and immunohistochemical staining indicated that BRE was strongly expressed in the zona glomerulosa of the adrenal cortex, which synthesizes and secretes the mineralocorticoid hormones. It is also highly expressed in the glial and neuronal cells of the brain and in the round spermatids, Sertoli cells, and Ledig cells of the testis, all of which are associated with steroid hormones and/or TNF synthesis. However, BRE expression was downregulated in human adrenal adenoma and pheochromocytoma, whereas its expression was enhanced in abnormal adrenal tissues of rats chronically treated with nitrate or nitrite. These data, taken together, indicate that the expression of BRE is apparently associated with steroids and/or TNF production and the regulation of endocrine functions. BRE may play an important role in the endocrine and immune system, such as the cytokineendocrine interaction of the adrenal gland. (J Histochem Cytochem 49:491499, 2001)
Key Words: BRE, adrenal, steroid, stress response, neoplasia
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Introduction |
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Steroid hormones play important roles in stress responses and in the maintenance of homeostasis of the organism. Steroids are synthesized mainly in specialized steroidogenic cells in the adrenal gland, in the ovary and placenta of females, and in the testis of males. The adrenal glucocorticoids and mineralocorticoids are important for carbohydrate metabolism, stress management, and salt balance (B.
We were interested in genes responsive to DNA damage and oncogenesis (B induced by TNF (
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Materials and Methods |
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Sample Preparation
Samples of human tissues were obtained from the Department of Pathology at Prince of Wales Hospital (Shatin, Hong Kong), and the pathology of the samples was identified by one of us (FMM Lai). Rat samples were from male SpragueDawley rats treated with or without 50 mg/liter of sodium nitrate or sodium nitrite in their drinking water for a period of 4 weeks to induce accumulation of lipid droplets in their adrenal glands (
Plasmid Preparation
The full-length BRE gene was amplified from a recombinant phage of gt11 as template (
Dot-blotting and Northern Blotting Analysis of mRNA
Dot-blotting and Northern blotting membranes were purchased from a commercial source (Clontech; Palo Alto, CA). The RNA master blot was used as a screening tool for the expression level of BRE mRNA in different human tissues. The multiple-tissue Northern blot was used to confirm the dot-blotting results. All hybridization steps were processed as described (-32P]-dCTP, using the Random Primers DNA Labeling Systems (Gibco/BRL; Rockville, MD). After prehybridization with the reagent provided by the manufacturer at 65C for 2 hr, the membranes were incubated with fresh hybridization buffer containing the labeled BRE probe and denaturedsonicated salmon sperm DNA and were hybridized overnight at 65C. The membranes were then washed several times with washing solution and exposed to X-ray films. To re-use the membranes, the labeled probe was stripped by washing the membranes twice for 20 min each with 0.5% SDS at 95C and rehybridized with the control gene ß-actin.
Preparation of BRE-specific Transcript Probes for ISH
To synthesize the labeled transcripts for RNA hybridization, the plasmid DNA of BRE was treated with restriction enzymes that created either a 5' overhang or a 3' overhang of the linearized vector, which were used as templates to synthesize the sense or antisense RNA probes. A digoxigenin (DIG)-labeled antisense RNA probe was obtained using an Nco I-digested BRE template, SP6 RNA polymerase, and with a DIG-RNA labeling kit from BoehringerMannheim (Mannheim, Germany) as described (
In Situ Hybridization
The method was essentially the same as described by us recently for the PML gene (
Preparation of BRE Antibody
To generate BRE antibody, a recombinant polypeptide of BRE from codon 38 to 226 was expressed in E. coli using the pRSET-B vector. The 30-kD BRE recombinant protein was purified through an Ni+ column from the bacterial lysate. The antibody was produced in rabbits and subsequently purified through affinity chromatography of the recombinant BRE protein on a fee-for-service basis by Research Genetics Laboratory (Huntsville, AL). The titer of the antibody was determined to be positive at over 200,000-fold dilution with an ELISA assay. The antibody was also characterized by Western blotting and immunohistochemical staining, indicating specific interaction with the BRE protein expressed in HeLa and 293 cells after transfection with the BRE cDNA.
Immunohistochemical Staining
Rat or human tissues were fixed in 10% formalin in PBS for 20 hr before dehydration in graded ethanol and embedding in Paraplast. Tissue sections of 5 µm were cut and mounted on silanized slides. After dewaxing with xylene and rehydration with an alcohol gradient, the slides were boiled in 10 mM sodium citrate buffer, pH 6.8, for 10 min in a microwave oven. The samples were then treated with 3% hydrogen peroxide in PBS for 10 min to quench endogenous peroxidase activity. After blocking with 5% rabbit serum for 20 min to reduce the nonspecific binding, the sections were incubated for 1 hr with rabbit anti-BRE antibody diluted at 1:800, then for 20 min with biotinylated goat anti-rabbit IgG (Zymed; San Francisco, CA), and then for 20 min with HRPstreptavidin (Zymed). The reactivity of the BRE antibody was visualized by incubating the slides with DABhydrogen peroxide solution from a DAB detection kit (Zymed). Finally, the sections were counterstained with hematoxylin, dehydrated, and mounted.
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Results |
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Differential Expression of BRE in Various Human Tissues Using the Dot-blotting Hybridization Assay
To rapidly determine the expression of this new gene, BRE, in different tissues, we used a commercially available mRNA dot-blot membrane, the RNA MasterBlot from Clontech, to examine BRE expression in a large number of tissues. As shown in Fig 1, 50 different human tissue samples were analyzed. The expression of BRE was strongest in adrenal gland, followed by the heart, pituitary gland, spleen, skeletal muscle, occipital lobe, and putamen. Moderate expression was found in the whole brain, testis, and ovary, and low expression was found in all fetal tissues, appendix, cerebellum, thymus, peripheral leukocytes, trachea, and placenta.
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Differential Expression of BRE in Various Normal Human Tissues Using the Northern Blotting Analysis
To rule out nonspecific hybridization in the above dot-blotting assay, Northern blotting was then performed on some of the tissue mRNA, particularly that of the adrenal gland. To avoid differences created in sample preparation, a commercially available Northern blot membrane, a Multiple-Tissue Northern Blot, was purchased and analyzed (Clontech). As shown in Fig 2, Northern blotting analysis of eight different human tissues indicated that only one major species of mRNA at 1.9 kb was hybridized to the BRE probe (Fig 2A), similar to the results published previously by us (
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ISH of BRE mRNA and IHC Staining of BRE in Various Rat Organs
To elucidate the cell types or specific regions of the various tissues that expressed BRE, ISH was performed. Because the human BRE gene shares more than 90% homology with the BRE cDNA fragments from rodent, as shown by the NCBI-Genbank data search, rat organs were used that are readily available. Fig 3A and Fig 3B show the ISH of the human BRE in the adrenal gland of the rat with the antisense probe. Strong expression of BRE mRNA (dark purple staining) was found in the cytoplasm of cells at the zona glomerulosa (ZG) and moderate expression in the zona fasciculata (ZF) of the adrenal cortex (c). The adrenal medulla (m) also showed considerable expression. In contrast, the control hybridization with the BRE sense probe in the same adrenal tissue showed no staining (Fig 3C). The hybridization was confirmed by IHC staining with the BRE antibody (Fig 3D and Fig 3E), which also showed strong reaction in the ZG (dark brown staining). BRE protein was localized mostly in the cytoplasm and in the perinuclear regions of cells. However, treatment with pre-immune serum produced no detectable reaction, indicating that the staining was specific (Fig 3F). In the rat brain, ISH with BRE antisense probe gave positive results in the glial cells (Fig 3G, arrowhead). In the rat testis, the immature spermatocytes and the Sertoli cells stained strongly with the BRE antisense probe (Fig 3H, arrow), while the Leydig cells were weakly positive (Fig 3H, arrowhead). In contrast, only the bile duct cells in rat liver showed strongly positive signal (Fig 3I, arrowhead), while the hepatocytes showed weak hybridization (Fig 3I, arrow). The IHC stainings for BRE (dark brown) of the latter three tissues are shown in Fig 3J3L respectively. In brain, the cytoplasm of both the glial cells and the pyramidal neuronal cells showed positivity. In testis, the interstitial Leydig cells, which are the androgen-producing cells, showed strong positive signal for BRE protein (Fig 3K, arrowhead). In liver, the hepatocytes showed weak but detectable BRE expression (Fig 3L).
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Decreased Expression of BRE mRNA in Adrenal Tumors
To determine if BRE expression may be altered in neoplastic tissues of the adrenal gland, human adrenal adenomas and pheochromocytomas were analyzed. Fig 4A and Fig 4D show the ISH of non-tumorous human adrenal gland, which showed strong expression of BRE mRNA. Adrenal adenomas were tumors of the adrenal cortex, which were from patients with primary hyperaldosteronism (Conn's syndrome). These neoplasias showed decreased or undetectable expression of the BRE mRNA in the tumor cells (T) as compared to the adjacent non-tumorous cells (Fig 4B and Fig 4E, arrowhead) in all of the samples examined (n = 5). Similarly, in pheochromocytomas, tumors of the adrenal medulla, there was decreased or undetectable expression of BRE mRNA in all of the samples (n = 4) analyzed (Fig 4C and Fig 4F).
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Enhanced Expression of BRE mRNA in Adrenal Glands of Rats Treated with Nitrate or Nitrite
It has been previously documented that inorganic nitrate inhibits gonadotropin-stimulated steroidogenesis in mouse Leydig tumor cells (
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Discussion |
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Although BRE had previously been found to bind to TNFR-1, its precise role in vivo is still unknown. Here we found increased expression of BRE mRNA and protein in several steroid hormone-producing organs, including the adrenal gland, testis, ovary, and brain. ISH and immunohistochemical staining identified the cell types with strong BRE signals, including the cells in the zona glomerulosa of the adrenal gland. In contrast, BRE mRNA and protein in zona fasiculata and zona reticulata was much reduced. Cells at the zona glomerulosa of the adrenal synthesize and secrete mineralocorticoid hormones, such as aldosterone (-helical segments, which were previously postulated to represent putative phospholipid interaction sites and mitochondria targeting. It also contains peroxisome localization signals (SRL-XXX-SRL) consisting of two tripeptides of SRL at codons 130 and 136 as well as a two-step for peptides (RXøXXS) at codons 131136 for mitochondrial protein. These motifs were apparently similar to those of the sterol carrier protein-2 (SCP2) (
(
However, the majority of adrenal adenoma represents Conn's syndrome, with hyperaldosteronism and low plasma renin (
It is interesting to note that several cytokines, including TNF, are known to stimulate the hypothalamopituitary corticotropin-releasing hormone (CRH/ACTH system), thereby evoking a secretory response by the adrenal cortex ( are synthesized in the adrenal gland by parenchymal cells and macrophages. The release of TNF is regulated by the main agonists of steroid hormone secretion, including ACTH and angiotensin-II, and bacterial endotoxins. TNF and IL-1 directly inhibit aldosterone secretion by zona glomerulosa cells, whereas IL-6 enhances it. In addition, TNF depresses glucocorticoid synthesis. There is evidence that local immuneendocrine interactions may play an important role in modulating adrenal responses to inflammation and to immune challenges and stresses. Here, we showed that the expression of BRE is in the zona glomerulosa, which is the exclusive site for synthesis and release of TNF. In this context, BRE may play an important role in regulating the cytokineendocrine interaction in the adrenal gland.
Because the glial cells are the supportive cells in the brain, they are rapidly activated in response to injury, inflammation, neurodegeneration, and infection. The microglial cells, which are the resident macrophages, produce and release TNF ( is secreted by the round spermatids in the seminiferous tubules and by macrophages in the interstitial tissues (
In view of the complexity and the diverse roles of steroids, TNF, and other signaling molecules in cell survival, apoptosis, growth, and differentiation, it is not surprising that BRE is expressed differently in different tissues and cell types and that it may have differential effects on cells. We are now generating transgenic and knockout models for analyzing the in vivo effect of BRE, and therefore, our results may provide clues to the organs and the phenotypes to be identified.
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Acknowledgments |
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Supported in part by CUHK-UGC grant 2040519, RGC-earmark grant CUHK 4279/97M, and a grant from National Science Council, Taiwan, awarded to JYH Chan, and by a UGC grant awarded to NS Panesar.
Received for publication November 8, 2000; accepted November 8, 2000.
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