Altered Expression Patterns of Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in the Adrenal Cortex
Institute of Basic Medical Sciences, University of Tsukuba, Ibaraki, Japan (WWW,HK); Kensei General Hospital, Ibaraki, Japan (MF); and Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan (YY,HS,HH,TY,EU,YA)
Correspondence to: Hiroshi Kamma, Institute of Basic Medical Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki, 305-8575, Japan. E-mail: hkamma{at}md.tsukuba.ac.jp
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Summary |
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(J Histochem Cytochem 53:487495, 2005)
Key Words: hnRNP A2/B1 alternative splicing adrenal cortex ACTH hormonal activity
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Introduction |
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The role of alternative splicing in responsiveness to ACTH is important (Leo et al. 2002). In cortical cells, ACTH stimulation is transduced mainly through a cyclic AMP-dependent signaling pathway, which regulates the transcription of target genes that have a consensus cAMP response element (CRE) in their promoters. CRE-binding transcription factors, such as CREM (CRE modulator) and CREB (CRE-binding protein), play important roles in the final step of this pathway (Lalli and Sassone-Corsi 1994
). CREM and CREB function either as repressors or as activators, which are controlled by alternative splicing or alternative transcriptional start sites of these genes (Yin et al. 1995
). Furthermore, ACTH, angiotensin II receptor, and some enzymes related to hormone production have several splicing variants that act differently (Yang et al. 1995
; Martin et al. 2001
; Rotzer et al. 2001
). These variants suggest that alternative splicing is an important regulating mechanism in hormonogenesis, although this mechanism is poorly understood.
The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a family of nucleic acidbinding proteins and comprise 24 different polypeptides termed hnRNPA1 through hnRNPU (Pinol-Roma et al. 1988). Although the functions of hnRNPs are diverse, they are implicated in most stages of mRNA metabolism, including transcription, splicing, nucleocytoplasmic transport, stability, and localization (Krecic and Swanson 1999
). The basic A/B group of hnRNPs, such as A1 and A2/B1 proteins, antagonizes the in vitro alternative splicing activity of splicing factors SF2/ASF or SC35 and influences 5' splice-site selection and exon skipping (Mayeda et al. 1994
). Previously, we reported that A2 and B1 proteins show an interesting expression pattern in murine adrenals (Kamma et al. 1995
). The A/B group of hnRNPs is being considered as an important candidate for regulation of hormonal function in alternative splicing. However, the adrenal expression of these has not yet been studied. On the other hand, there have been many reports that the B1 protein is overexpressed specifically in tumors of several organs (Hamasaki et al. 2001
; Zhou et al. 2001
).
The final goal of our study is to elucidate the alternative splicing mechanism, which is related to the adjustment of cytodifferentiation and steroidogenesis regulation in the adrenal cortex. In this study, the histological expression of the A2 and B1 proteins was examined in the adrenal cortex and functional adrenocortical tumors. Interesting expression patterns were found related to the functional differentiation of the adrenal cortex.
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Materials and Methods |
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Cell Cultures
A human adrenocortical carcinoma cell line, H295R, was purchased from ATCC (Rockville, MD). It was grown in DMEM/Ham's F-12 (1:1) supplemented with 2.5% fetal calf serum, 2% Ultroser G, 2 mmol/L glutamine, 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 5 ng/ml linoleic acid, 1.25 mg/ml bovine serum albumin, 50 U/ml penicillin, and 50 mg/ml streptomycin (Sigma; St Louis, MO). HeLa cell line (JW 36) was utilized as a control. Both cells were cultured at 37C in a 5% CO2 incubator.
Preparation of Antibodies and Immunohistochemistry
Anti-hnRNP A2 and B1 monoclonal antibodies, 4G8 and 2B2, were generated previously in our laboratory (Kamma et al. 2001). All antibodies were in the form of ascites fluid or protein-Apurified antibody, and their titers were adjusted by normalization to immunostaining of HeLa cells.
Paraffin tissue sections were cut into 4-µm-thick slices, deparaffinized, and heated in 0.01 M citrate buffer (pH 6.0) by microwave irradiation for antigenic retrieval. The sections were preincubated with 3% BSA in phosphate-buffered saline (PBS) to diminish the background and then incubated with antibodies for 1 hr at room temperature. Bound antibodies were detected by a peroxidase-labeled polymer-conjugated anti-mouse antibody (EnVision+; DAKO, Fort Collins, CO), and reacted with 0.025% 3,3'-diaminobenzidine tetrahydrochloride. Hematoxylin was utilized for counterstaining. To objectively estimate the expression pattern of the A2 and B1 proteins, the positive cells for 4G8 and 2B2 were morphometrically analyzed as described previously (Wu et al. 2002) in the seven normal adrenal tissues. The results were presented as the mean ± SD. Statistical analysis was evaluated by Student's t-test. Differences were considered significant at p<0.05.
Gel Electrophoresis and Western Blotting
The frozen samples of normal adrenal tissues and adrenocortical adenomas were crushed and homogenized in sodium dodecyl sulfate (SDS) sample buffer. The protein concentration of each sample was measured by a protein assay kit (Bio-Rad; Hercules, CA). Protein from each sample (50 µg) was separated by electrophoresis on 12.5% SDS-polyacrylamide gels. Proteins were transferred to nitrocellulose (PROTRAN; Schleicher and Schuell, Keene, NH) and reacted with monoclonal antibodies 4G8 and 2B2. Bound antibodies were detected with peroxidase-conjugated anti-mouse antibodies from sheep (Amersham; Buckinghamshire, UK) and the ECL system (Amersham). To quantitatively estimate the expression of A2 and B1, SDS tissue samples were adjusted to contain 50 µg of protein/lane, and the blotting result images were analyzed by a densitometer (GS-700 Imaging Densitometer; Bio-Rad).
ACTH Stimulation of H295R Cells
H295R cells were washed twice with PBS and then cultivated in a medium containing ACTH (Sigma), ranging from 0 M to 107 M, or forskolin (Sigma), ranging from 0 µM to 20 µM. The cells were harvested from 0 to 72 hr at 12-hr timepoints, counted, and prepared at 3 x 106 cells with 300-µl SDS sample buffer for each timepoint. Concurrently, the culture supernatants were collected for estimating hormone production by radioimmunoassay, including cortisol and dehydroepiandrosterone sulfate (DHEA-S), which is stably produced from the H295R cells and is recognized as an adrenal sexual steroid precursor (Gell et al. 1998). The experiments were performed three times.
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Results |
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Discussion |
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Interestingly, B1 expression was higher in clear-type tumor cells than in compact-type tumor cells, which was reverse to B1 expression in the normal adrenal cortex. Electron microscopic studies of cortical tumors have revealed that clear-type tumor cells have abundant smooth endoplasmic reticulum and mitochondria (Eto et al. 1979; Gazdar et al. 1990
). This suggests that clear-type tumor cells have high hormone-producing activities, unlike normal cortical cells.
In addition, non-tumor parts of the adrenal cortex in cortisol-producing tumors had less B1 protein than in normal adrenals, whereas those in the aldosterone-producing tumors had more B1 protein. The cortical cells in vivo function under a complicated feedback mechanism, which is controlled mainly by ACTH secreted from the pituitary gland. In patients with cortisol-producing tumors, the ACTH serum level is usually low and the non-tumor part is histologically atrophic, indicating low hormonal activity. On the other hand, in patients with aldosterone-producing tumors, ACTH secretion is not suppressed and the non-tumor parts are normal or hyperplastic (Murakoshi et al. 1995). This also suggests the correlation of B1 expression in the cortical cells to the hormone-producing activity responding to ACTH.
A simple experimental model of cortical hormonogenesis is necessary to show the relationship between B1 expression and ACTH stimulation. The H295R cell line has receptors for ACTH and retains the ability to produce mainly androgen in response to ACTH (Gazdar et al. 1990; Mountjoy et al. 1994
), although the number of receptors is less than in normal cortical cells. ACTH increases intracellular cAMP production and hence activates the PKA pathway and CRE-binding transcription factors. In this experiment, H295R cells were utilized to reproduce the adrenocortical hormonogenesis controlled by ACTH. After ACTH stimulation, H295R cells showed biphasic patterns of DHEA-S secretion. This biphasic reaction to ACTH has been reported previously (Jefcoate et al. 1987
; Le Roy et al. 2000
) and was explained by the biphasic up- and downregulation of ACTH receptors in H295R cells (Fassnacht et al. 1998
,2000
). This was reproduced also in a forskolin stimulation experiment (data not shown). In this experiment, B1 also showed a dramatic biphasic expression pattern that almost paralleled that of DHEA-S secretion, whereas the change of A2 was less dramatic, although it showed the same biphasic pattern. The maximum point of the biphasic change of B1 expression apparently precedes hormonal production. After prolonged stimulation (72 hr) with ACTH (Figure 7), H29R cells showed a notable decrease of B1 expression in a dose-dependent manner. However, A2 did not exhibit obvious change. The decrease of B1 may reflect the low responsiveness to ACTH, which has been observed under a distinct experimental condition of prolonged ACTH stimulation, representing an in vivo shock state (Slawik et al. 2004
). B1 protein may be involved in an intercellular feedback mechanism via the cAMP pathway during chronic continuous stimulation with ACTH. A2 and B1 are transcribed from an identical gene (Kozu et al. 1995
). The change of the ratio between the A2 and B1 proteins may be caused by alternative splicing or posttranslational mechanisms. Preliminary results of quantitative RT-PCR in H295 cells stimulated by ACTH showed no apparent change in the A2/B1 mRNA ratio (data not shown). It is assumed that the dissociation between the A2 and B1 proteins is caused by differences in posttranslational metabolism.
In conclusion, we have shown that B1 protein expression is variable in the adrenal cortex, especially in the zona fasciculata-reticularis, and correlates with the producing activity of cortical hormones responding to ACTH stimulation. An additional goal will be to elucidate the alternative splicing mechanism in the adrenal cortex. Further in vivo studies are necessary to clarify the relationship between B1 protein expression and the alternative splicing pattern of regulatory genes such as ACTH receptors, CRE-binding transcription factors, and enzymes involved in steroidogenesis.
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Footnotes |
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