Ad4BP in the Human Adrenal Cortex and Its Disorders
HIRONOBU SASANO, SOUICHIROU SHIZAWA, TAKASHI SUZUKI,
KAZUTO TAKAYAMA, TAKAO FUKAYA, KEN-ICHIROU MOROHASHI, AND HIROSHI NAGURA
Departments of Pathology (H.S., S.S., T.S., H.N.) and Obstetrics and Gynecology (K.T., T.F.), Tohoku University School of Medicine, Sendai, Japan 980; and Department of Molecular Biology (K.M.), Graduate School of Medical Science, Kyushyu University, Higashi-ku, Fukuoka, Japan 812
ABSTRACT
Ad4BP, a zinc finger DNA-binding protein, is a transcription factor that regulates the expression of the steroidogenic P450 genes. We performed immunoblotting and immunohistochemistry of Ad4BP in 34 human adrenal cortex specimens, which included adrenocortical adenomas and carcinomas. Immunoblotting revealed a single band of 53K, corresponding to the mol wt of Ad4BP. The immunohistochem- ical studies demonstrated that Ad4BP immunoreactivity was present exclusively in the nuclei of nearly all of the adrenocortical parenchy- mal cells in both the normal and the pathological human adrenal
specimens. Ad4BP was immunostained with equal intensity and fre- quency among the different cell types. Ad4BP immunoreactivity was also observed in areas of marked degenerative changes, such as li- pomyelomatous lesions, and in poorly differentiated carcinoma cells. These results suggest a close association of Ad4BP expression with the biological phenotype of adrenocortical parenchymal cells. Ad4BP therefore seems to play important roles in the induction and main- tenance of the transcription of all steroidogenic P450 genes in human adrenocortical cells, even after malignant transformation. (J Clin Endocrinol Metab 80: 2378-2380,1995)
R ECENTLY, examination of the promoter region of the bovine CYP11B gene revealed the presence of an adrenal(Ad)4 site, which is required for the full response to complementary AMP and the cell-specific transcriptional ac- tivity of the steroidogenic enzyme genes (1). This Ad4 site or steroidogenic factor (SF)-1 binding site was detected subse- quently in all of the other P450 genes involved in steroido- genesis (2-4). These results indicated functional significance of this Ad4 or SF-1 binding site in steroidogenesis, regardless of the products that were synthesized. In addition, an orphan nuclear receptor protein that binds to the Ad4 site, termed Ad4BP or SF-1, was purified from the bovine adrenal cortex (2, 5, 6). Subsequent studies of expression of Ad4BP in rats demonstrated that Ad4BP is expressed only in the steroido- genic tissues, with the exception of gonadotropin-producing cells in the pituitary glands of mice (7). By using targeted gene disruption, Luo et al. (8) recently produced mice that were homozygously deficient in SF-1 (Ad4BP). These SF-1- or Ad4BP-null mice survived normally in utero, but all died by postnatal day 8 because of adrenocortical insufficiency; both the adrenal and the gonads were not formed in these animals (8). Results of these studies demonstrated the es- sential role of the gene encoding Ad4BP in the embryonic differentiation and biological function of the primary steroi- dogenic tissues, including adrenal cortex.
It is well known that human corticosteroid secretion and metabolism are different from those in other mammals, in- cluding rats. However, the regulation of human adrenocor- tical steroidogenesis has not been well studied compared with that of various experimental animals. In addition, little
is known about the regulation of steroidogenesis at the cellular level in human adrenocortical disorders that are associated with abnormal corticosteroidogenesis. Furthermore, different cell types in the human adrenal, including adrenocortical neo- plasms, synthesize or metabolize corticosteroids differently. Therefore, in order to obtain a better understanding of both normal and pathological human adrenocortical steroidogene- sis, we initially examined by immunoblotting whether or not Ad4BP is expressed in normal and pathological specimens of the human adrenal cortex. We then performed immunohisto- chemistry of Ad4BP in 34 human adrenal specimens in order to determine in which cell types Ad4BP is expressed and whether the rigid specificity of the expression of Ad4BP that has been reported in mouse (9) and rat (10) adrenals is also observed in the human adrenal cortex, especially in adrenocortical disorders.
Material and Methods
Human adrenal
Thirty-four human adrenal specimens were examined in this study: 15 nonpathological human adrenal specimens obtained from radical nephrectomy caused by renal cell or transitional cell carcinoma; 6 al- dosteronomas; 5 adrenocortical adenomas associated with no clinical hormonal abnormalities or nonfunctioning adenomas; 3 Cushing’s ad- enomas; and 5 adrenocortical carcinomas. Adrenocortical carcinomas were histopathologically diagnosed as determined by the criteria of Weiss (11).
The specimens were fixed in 8% paraformaldehyde, pH 7.4, at 4 C for 18 h immediately after their surgical removal. After fixation, the specimens were embedded in paraffin wax.
Primary antibodies
The purification of Ad4BP and the subsequent production of a rabbit polyclonal antibody against Ad4BP and its characteristics have been described in detail by Morohashi et al. (10).
Immunoblot analysis
Immunoblot analysis was performed on two normal adrenals, on two patients with adenocortical carcinoma, and on five adenoma specimens from those patients (two aldosteronomas, two nonfunctioning adeno- mas, and one Cushing’s adenoma) with their corresponding nonneo- plastic adjacent adrenal glands. The tissues were homogenized in lysis buffer containing 50 mmol Tris-HCI (pH 7.4) and 2% sodium dodecyl sulfate, followed by sonication to disrupt DNA. The protein concentra- tions were determined on the basis of the method described by Kalb and Bernlohr (12). After the proteins in the samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, they were blotted onto a nitrocellulose filter. The filter was processed for the detection of Ad4BP with previously described antiserum to bovine Ad4BP. ECL Western blot reagents (Amersham, Arlington Heights, IL) were used for the detection of signals. Rat ovaries were employed as a positive control for immunostaining.
Immunohistochemistry
The immunohistochemical procedures were performed on 2.5-pum thick sections mounted on poly-L-lysine-coated slides using the biotin- streptavidin amplified technique with a Histofine immunostaining kit (Nichirei, Tokyo, Japan). The staining procedure was performed as follows: 1) routine deparaffinization; 2) inactivation of endogenous per- oxidase activity with 0.3% H2O2 in methyl alcohol for 30 min at 23 C; 3) blocking with 1% goat serum for 45 min at 23 C; 4) incubation with the primary antibody at 4 ℃ for 18 h; 5) incubation with biotinylated goat antirabbit antibody for 30 min at 23 C; 6) incubation with peroxidase- conjugated streptavidin for 30 min at 23 C; 7) colorimetric reaction with a solution containing 0.05% Tris-HCL, pH 7.6, 0.66 mol/L 3.3’-diami- nobenzidine, and 2 mol/L H2O2; and 8) counterstaining with 1% methyl- green. For a positive control of immunostaining, rat ovary and adrenal were employed. For a negative control, the antibody was preincubated with an excessive amount of purified Ad4BP or with 0.01 mol/L phos- phate-buffered saline instead of the primary antibody for 18 h at 4 C.
Results
Immunoblotting
Results are shown in Fig 1. Immunoblotting for Ad4BP demonstrated a single band of 53K in all of the human ad- renal samples that were examined, which corresponds to the mol wt of Ad4BP (2). Rat ovary, which was employed as a positive control, also demonstrated a single band of 53K.
Rat ovary
Normal
Cushing’s adenoma tumor adrenal
aldosteronoma NF carcinoma
66.2
tumor adrenal
45.0
31.0
21.5
Immunohistochemistry
Immunoreactivity for Ad4BP indicated that it was local- ized exclusively in the nuclei (Figs. 2, A, B, and C). Immu- noreactivity was not observed in the tissue sections incu- bated with preabsorbed rabbit serum or 0.01 mol/L phosphate-buffered saline (Fig. 2D).
Normal adrenal. Ad4BP immunoreactivity was observed in more than 90% of the cortical parenchymal cells in the zonae glomerulosa, fasciculata, and reticularis but not in the cells of the capsule, sinucoidal lining cells, and medullary chro- maffin cells (Fig. 2A). Intracortical heterogeneity of Ad4BP expression was observed, but the immunointensity and the number of Ad4BP-positive cortical cells were not different among the zonae glomerulosa, fasciculata, and reticularis (Fig. 2A). Ad4BP immunoreactivity was also observed in cortical cells undergoing degeneration, including myeloli- pomatous changes.
Adenoma. Ad4BP immunoreactivity was observed in more than 90% of the adrenocortical tumor cells, including non- functioning adrenocortical adenoma (Fig. 2B) as well as in the adrenocortical parenchymal cells of adjacent, nonneoplastic adrenal glands, including the atrophic zona fasciculata-re- ticularis of Cushing’s adenomas. Intratumoral and intraad- renal heterogeneity of Ad4BP expression was observed, but the ratio of the Ad4BP-positive cells and the relative Ad4BP immunointensity were not different among the aldosterono- mas, nonfunctioning adenomas, and Cushing’s adenomas. Among different types of cortical adenoma cells, there were no differences in the relative Ad4BP immunointensity or in the number of Ad4BP positive cells.
Carcinoma. Ad4BP immunoreactivity was observed in more than 90% of the carcinoma cells in the cases examined (Fig. 2C). Ad4BP immunoreactivity was observed in carcinoma cells with relatively low nuclear grade, in well differentiated carcinoma cells (Fig. 2C), in carcinoma cells with bizarre nuclei, and in poorly differentiated carcinoma cells with equal frequency and immunointensity.
Discussion
Our present study demonstrates that Ad4BP is expressed in normal and abnormal human adrenal cortex by immu- noblotting. Using immunohistochemical techniques, a close relationship was shown between Ad4BP expression and cor- tical parenchymal cells in which steroidogenic enzymes were demonstrated (13). This result is consistent with the possible role of Ad4BP as the universal regulator of steroidogenic P450 gene transcription in both normal and neoplastic human corticosteroidogenesis.
Neither the relative Ad4BP immunointensity nor the num- ber of Ad4BP positive cells was different among disparate adrenocortical cell types, which have been known to express diverse kinds and amounts of steroidogenic enzymes (13). In addition, nuclear Ad4BP immunoreactivity was observed with equal frequency and intensity in the zonae fasciculata and reticularis of the attached and atrophied nonneoplastic adrenals of Cushing’s adenomas, in which the expression of
A
B
C
G
R
M
F
C
D
steroidogenic enzymes was thought to be below the level of detection by immunohistochemistry because of suppressed pituitary ACTH secretion (13). Therefore, Ad4BP is consid- ered to play a more constitutive role in human adrenal cor- ticosteroidogenesis, i.e. the initiation and maintenance of the transcription of all P450 genes, and the level of its expression does not necessarily correlate with that of the intracellular steroidogenic P450s.
Human adrenocortical cells undergo various biological changes, such as degenerative, hyperplastic, and neoplastic or malignant changes. The expression of steroidogenic en- zymes is maintained throughout the process of these cyto- logical changes (13). Our present study demonstrated that Ad4BP expression is also observed in cells undergoing these changes, which include myelolipomatous degeneration and malignant transformation. Therefore, expression of Ad4BP is considered to be essential for maintenance of the biological characteristics of adrenocortical cells even after malignant transformation. This observation is also consistent with an essential role of Ad4BP in the development of adrenal glands in other animals (8).
In summary, our study of the human adrenal and its dis- orders, including adrenocortical carcinoma, demonstrated a close association between Ad4BP expression and adrenocor- tical parenchymal cells.
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