Metallothionein-3 (MT-3) in the Human Adrenal Cortex and its Disorders
Saulo J. A. Felizola · Yasuhiro Nakamura · Yuki Arata ·
Kazue Ise . Fumitoshi Satoh . William E. Rainey .
Sanae Midorikawa · Shinichi Suzuki · Hironobu Sasano
Published online: 17 November 2013 C Springer Science+Business Media New York 2013
Abstract Metallothionein-3 (MT-3) is an intracellular, low molecular weight protein mainly distributed in the central nervous system but also in various peripheral organs and several types of human neoplasms. However, details of MT- 3 expression have not been examined in human adrenal cortex and its disorders. The mRNA levels of MT-3 were first eval- uated by quantitative RT-PCR (qPCR) in adrenocortical aldosterone-producing adenoma (APA: 11) and cortisol- producing adenoma (CPA: 14). In addition, MT-3 immuno- histochemistry was performed in non-pathological adrenal glands (NA: 19), idiopathic hyperaldosteronism (IHA: 10), APA (20), CPA (24), adjacent non-neoplastic adrenal glands of adenoma (AAG: 20), and adrenocortical carcinoma (ACC: 8). H295R cells were also treated with angiotensin-II or forskolin in a time-dependent manner, and the changes of MT-3 mRNA levels were evaluated by qPCR. Results of qPCR analysis demonstrated that MT-3 mRNA levels were significantly higher in APA than CPA (P=0.0004). MT-3
S. J. A. Felizola · Y. Nakamura . Y. Arata · K. Ise . H. Sasano Department of Pathology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8575, Japan
e-mail: yasu-naka@patholo2.med.tohoku.ac.jp
F. Satoh
Division of Nephrology, Endocrinology and Vascular Medicine, Tohoku University Hospital, Sendai, Japan
W. E. Rainey Department of Physiology and Medicine, University of Michigan, Ann Arbor, MI, USA
S. Midorikawa Department of Radiation Health Management, Fukushima Medical University, Fukushima, Japan
S. Suzuki
Department of Organ Regulatory Surgery, Fukushima Medical University, Fukushima, Japan
immunoreactivity was detected in the zona glomerulosa of NA, IHA, and AAG, as well as in APA, CPA, and ACC. When treated with angiotensin-II and forskolin, MT-3 mRNA levels reached a peak by 12 h in H295R cells, with signifi- cantly higher levels compared to control non-treated cells (P<0.01). The presence of MT-3 in the ZG of NA, IHA, and AAG, as well as APA may imply a role in the pathophysiol ogy of aldosterone-producing tissues.
Keywords Adrenal cortex · Zona glomerulosa · Adrenocortical adenoma · Aldosterone · Metallothionein · Immunohistochemistry
Introduction
Metallothioneins (MTs) are intracellular, low molecular weight proteins containing an array of conserved 20 cysteines and polynuclear metal-sulfur coordination sites formed by metal ions [1, 2]. Their most widely detected isoforms in mammals, MT-1 and MT-2, are rapidly induced in the liver by a wide range of metals, drugs, and inflammatory mediators [3]. In addition to MT-1 and MT-2, two tissue-specific MTs have been reported: MT-3 in the central nervous system (CNS) and MT-4 in the epithelial cells [4]. The CNS- specific isoform, MT-3, was reported to exert a growth inhib- itory action specifically on neuron cells, while MT-1 and MT- 2 have more diverse functions related to their thiolate cluster structure [3].
MT-3 was originally identified as a neural growth inhibi- tory factor which did suppress outgrowth of rat cortical neu- rons in brain extracts [5, 6]. In neurons, MT-3 regulates various lysosomal functions, and its absence results in reduc- tions of certain lysosomal enzymes, resulting in decreased autophagic flux [5]. MT-3 has been mainly detected in the CNS as above, but also reported to be expressed in various
peripheral human and rat organs and in several types of human tumors, including prostate, lung, breast, urothelial, and esoph- ageal carcinomas [7-14].
Of particular interest, recently published results of the gene expression profiles in aldosterone-producing adenoma (APA) and adjacent adrenal gland (AAG) showed transcripts with elevated MT-3 expression levels in APA versus AAG tissue [15]. However, details on MT-3 expression within the NA cortical zonation or other types of adrenocortical adenoma were not evaluated. Although MT-1/2 have been previously reported in the adrenal cortex [16], their function and tran- scriptional regulation is postulated to be different from that of MT-3, which is not induced by heavy metals like the former, and is suggested to be more involved in cell growth inhibition and metabolism control than the MT-1/2 isoforms [17-20].
Therefore, in this study, we evaluated the mRNA and protein expression of MT-3 in APA and cortisol-producing adenomas (CPA), as well as its immunoreactivity in the three layers of the non-pathological adrenal cortex (NAC), idiopathic aldosteronism (IHA), AAG, and adrenocortical carcinomas (ACC).
Materials and Methods
Human Adrenal Cases
Research protocols were approved by the ethics committees at Tohoku University Graduate School of Medicine (Sendai, Japan) and the Fukushima Medical University (Fukushima, Japan). For quantitative RT-PCR analysis (qPCR), adrenocor- tical neoplasms were obtained from Tohoku University Hos- pital (5 APA and 8 CPA) or Fukushima Medical University Hospital (6 APA and 6 CPA). The samples were snap-frozen and preserved in -80 ℃ conditions until use.
For immunohistochemical analysis, 19 NAC, 10 IHA, 52 adrenocortical tumor specimens (20 APA, 24 CPA, and 8 ACC), and 20 AAG were retrieved from the surgical pathol- ogy files of Tohoku University Hospital (Sendai, Japan). All ACC evaluated in our present study were considered non- hormone-producing tumors following the review of the charts of the patients.
RNA Isolation and qPCR
RNA isolation with subsequent cDNA production and qPCR technique were performed as previously reported [21]. The primer sequences used in this study were MT-3 forward 5’-TCA CCA CGT GCA GTA TCT CAA GAT ATT CAG-‘3, reverse 5’-ACC TAA GAC ATT GAG TGG AGA GTC CTA ACC-‘3; RPL13A forward 5’-CCT GGA GGA GAA GAG GAA AG-‘3, reverse 5’-TTG AGG ACC TCT GTG TAT TT-’ 3. The cDNA produced from a human brain specimen was
used as a positive control in qPCR experiments. RPL13A was used as an endogenous control gene in all qPCR experiments.
When tissue samples were analyzed, the relative gene expression was calculated as previously reported [21]. For the analysis of data derived from cell experiments, the relative gene expression was calculated by the AACt method as previously reported [22].
Immunohistochemical Analysis
Rabbit polyclonal antibody against human MT-3 was pur- chased from Sigma-Aldrich (St. Louis, MO, USA) and used at a 1:500 dilution. IHC technique was carried as previously described [21]. After completely reviewing the slides, immu- noreactivity of MT-3 in each zones of the adrenal cortex as well as in tumor specimens was evaluated by labeling index when immunoreactivity was located in nuclei through first selecting five representative high-power fields and counting 1, 000 cells among these fields. When immunoreactivity was detected in the cytoplasm, a semiquantitative evaluation was employed, as described previously in our groups [23]. All the evaluations above were independently and blindly performed by two of the authors (SJAF and YA), and the mean values were used for subsequent analysis.
Cell Culture
Human adrenocortical carcinoma cells, H295R, were cultured in DMEM/Eagle’s F12 medium (Invitrogen, Carlsbad, CA, USA) and supplemented with 10 % Cosmic Calf Serum (Hyclone Laboratories Inc., Nampa, ID, USA), 1 % penicillin/streptomycin (Invitrogen), and 0.01 % gentamycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in a 37 ℃ humidified atmosphere (5 % CO2).
H295R Cells Treatment and qPCR Analysis
H295R cells were transferred to 12 wells dishes in groups of 600,000 cells per well. After 24 h from passage, the medium was changed to DMEM/Eagle’s F12 medium supplemented containing 0.1 % Cosmic Calf Serum and media containing angiotensin-II (Tocris, Bristol, UK) (10 nM) or forskolin (FSK) (Tocris) (10 µM) were added after 48 h to different groups of the cells, each group comprising three wells. A basal group, to which vehicle was added, was used as a control in this experiment. RNA was extracted at 3, 6, 12, and 24 h time points (RNeasy Mini Kit, QIAGEN, Hilden, Germany). All the experiments were independently performed in triplicate.
Statistical Analysis
The data from the cell experiments and immunohistochemical analysis were analyzed using t tests or ANOVA post hoc tests
MT3 / RPL13A relative mRNA levels
0
05
1
.15
.2
.25
.3
.35
APA (n=11)
*
CPA (n=14)
with Bonferroni multiple correction. Data from human tissue qPCR analysis was evaluated using a t test, and P<0.05 was considered significant.
Results
qPCR Analysis of Human Tissues
MT-3 mRNA was detected in all the specimens of human adrenocortical tissues examined. The mRNA levels were
a
b
C
d
Cytoplasmic immunostaining (%) ®
**
*
Nuclear immunostaining (%)
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*
*
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40
20
20
10
0
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APA CPA ACC
APA CPA ACC
significantly higher in APA compared to CPA (P=0.0004) as summarized in Fig. 1.
Immunohistochemical Analysis
MT-3 immunoreactivity was detected relatively diffusely in APA and CPA (Fig. 2a, b) and either diffusely or focally in ACC (Fig. 2c). The status of MT-3 nuclear immunoreactivity was significantly higher in APA than CPA (P=0.0164) or ACC (P=0.0008), but no statistical difference was detected between CPA and ACC (Fig. 2d). However, ACC
demonstrated significantly higher cytoplasmic levels of MT- 3 than APA and CPA (P<0.0001), with no statistically sig- nificant differences detected in the status of MT-3 cytoplasmic immunoreactivity between these adenomas (Fig. 2e). MT-3 immunoreactivity was also detected in the zona glomerulosa (ZG) ofNA, IHA, and AAG. Marked nuclear and cytoplasmic immunostaining was detected in the ZG in contrast to inner adrenocortical zones (Fig. 3a-c). The adrenocortical cells in- volved in excessive aldosterone production (i.e., hyperplastic ZG in IHA and neoplastic cells in APA) were significantly associated with higher nuclear concentration of MT-3, but the
a
b
ZG
ZF
ZG
ZF
C
ZG
ZF
d
100
Nuclear immunostaining (%)
90
80
70
60
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0
T
ZG
ZF *
ZR *
ZG *
ZF *
ZR *
ZF
ZG
ZR **
*
**
**
NA
IHA
AAG
e
100
Cytoplasmic immunostaining (%)
90
80
70
60
50
40
30
20
10
T
T
T
T
0
ZG ZF ZR * *
**
ZG *
ZF
ZR
ZG ZF ZR
**
**
NA
IHA
AAG
status of MT-3 cytoplasmic immunoreactivity was significantly higher in the ZG of NA than that of IHA (P<0.05) (Fig. 3d, e).
H295R Cells Treated with AII or FSK and qPCR Analysis
The time course of H295R cells following the treatment with angiotensin-II (AII) and FSK was summarized in Fig. 4. MT-3 mRNA reached highest levels at 12 h of treatment steeply going down after that time-point, indicating its involvement in the mid to relatively final stages of steroidogenic pathways of the cells. With FSK, MT-3 mRNA levels increased up to twofold relative to basal level, while with AII, up to 1.5-fold at its peak (P<0.01 AII vs. basal, FSK vs. basal, and AII vs. FSK were marked by correspondent asterisks in Fig. 4).
2.25
MT3 relative mRNA (fold)
2
1.75+
1.5
1.25+
1
3h 6h
12h
24h
**
*
**
Basal
All
Fsk
Discussion
Results of our present study demonstrated that MT-3 immu- noreactivity was detected in the ZG of NAC, IHA, and AAG as well as in APA and CPA both at protein and mRNA levels and was also detected at the protein level in ACC. In addition, results of in vitro analysis also demonstrated the presence of MT-3 mRNA at the basal levels and its treatment-dependent changes after AII and FSK stimulation in the 24-h time course.
The expression of neuron-specific proteins in APA and the ZG cells of human adrenocortical tissues has been reported including synaptophysin, neuronal cell adhesion molecule, serotonin-receptor 4, metabotropic glutamate receptor 3, and others [24-26], indicating the possible common regulatory processes between neurons and aldosterone-producing cells. Adrenocortical carcinomas have also been demonstrated to express neurofilament immunoreactivity [27]. It has been recently reported that the ZG cells are electrically excitable with increased levels of T-type Ca2+ channels, which are also present in neurons [28]. The precise functions of these neuron- specific proteins in adrenocortical aldosterone-producing cells have remained unclear; however, results of our present study indicated that MT-3 also represents one of various neural proteins expressed by these adrenocortical cells.
MT-3 has been previously reported in other peripheral tissues and tumors. It has been previously detected in human prostate and kidney as well as reproductive organs of rat; however, its function in these tissues has remained unknown [7, 8, 10]. This protein has also been reported to be expressed in CNS tumors (i.e., ependymoma), pituitary tumors (i.e., ACTH-producing adenoma and nonfunctioning adenoma), as well as human urothelial, prostatic, and breast carcinomas and corresponding cell lines; as reported, although not indis- pensable for malignant transformation, MT-3 presence may increase drug resistance or indicate a higher tumor grade, depending on the neoplasm [7, 13, 29-31]. In the CNS, MT- 3 is suggested to form a complex with proteins like the heat shock proteins 70 and 90 or with G protein Rab3A, which might enable MT-3 to inhibit the toxic effects of nitric oxide (NO) in neurons [18]. However, this possible functional role has not been confirmed in peripheral tissues.
In this study, we detected the nuclear and cytoplasmic localization of MT-3 protein in human adrenal cortex and its disorders, especially aldosterone-producing cortical cells. The presence of MT-3 in both nuclear and cytoplasmic compo- nents was previously reported in pediatric ependymomas [30], but the biological significance of MT-3 in different subcellular components has remained unknown. Between nuclear and cytoplasmic MT-3, the former was significantly associated with aldosterone-producing cells, especially in the ZG of IHA. However, MT-3 cytoplasmic immunoreactivity was more pronounced in the ZG of NA. Therefore, we suggest that the subcellular localization of MT-3 protein could be
related to excessive aldosterone production in the ZG, which may be clarified by further investigations. Different from ZG cells, however, adrenocortical adenoma cells had very low levels of nuclear and cytoplasmic MT-3 comparable to those detected in the zona fasciculata (ZF) despite excessive aldo- sterone production in these tumor cells. We believe that this may be explained by the fact that about 80 % of adrenal adenoma cases are formed by ZF-like cells, which predomi- nate in those tumors [32, 33]. The difference in MT-3 levels between adenoma types again suggests the role of that protein in pathological aldosterone-producing tissues, as significantly higher amounts of MT-3 at both mRNA and protein levels were detected in APA compared to CPA. The significantly higher amounts of cytoplasmic MT-3 in ACC, in contrast to benign tumors, indicated that MT-3 could play a role in biological behavior of adrenocortical malignancy, which has also been reported in other human malignancies [7, 11-14, 31].
As described above, the regulation of MT-3 in the CNS has been reported to be related to heat shock proteins 70 and 90, and a relationship to NO toxicity inhibition has been pro- posed, but its regulatory mechanisms still remain to be clari- fied [18]. MT-3 has recently been reported to be functionally related to the phosphatidylinositol-3 kinase (PI3K)/AKT path- way through which MT-3 could provide neural protection by exerting antiapoptotic effects [34]. This same PI3K/AKT pathway has been demonstrated to be over-activated in APA and IHA compared to normal ZG with positive correlation between the plasma levels of aldosterone and p-AKT, and the functional correlation to PI3K/AKT could provide the func- tional correlation between MT-3 status and hyperaldosteronism [35]. Results of the time-course experiment in H295R adreno- cortical cells demonstrated that MT-3 mRNA levels were con- trolled by both AII and FSK and the respective pathways. Romero et al., Nogueira et al., and others reported that on adrenal cortex and aldosterone-related research the mRNA variations in vitro are expected to reflect what happens in vivo [22, 25, 26, 28, 36-38], and thus, it is considered that AII and FSK may control MT-3 protein levels in adrenocortical tissues. FSK is known to induce the cAMP pathway, leading to an increase in CYP11B1/B2 expression levels with subsequent corticosteroid production in the adrenal cortex [37], while AII activates phospholipase C through G protein subunit q/11 (Gq/11), as well as several other signaling molecules, in- cluding protein kinase C, calcium/calmodulin-dependent kinases, mitogen-activated protein kinase kinase-extracel- lular signal-regulated kinase 1 and 2 (MEK1/2-ERK1/2), src-family kinases, ras/raf kinases (RAS/RAF), and Janus kinase (JAK)/signal transduction and activators of tran- scription [38]. These intricate pathways and numerous regulatory factors indicate that any possible mechanisms by which MT-3 may act under the influence of cAMP or AII are rather complex processes and should require fur- ther research for clarification.
Conclusion
As its immunolocalization and mRNA patterns suggest, MT-3 may have a role in aldosterone-producing adrenocortical cells, as well as in adrenocortical carcinoma. However, further studies are necessary to clarify its possible functions in adre- nocortical tissues.
Acknowledgments This work was partly supported by the Takeda Science Foundation and partly supported by the grant for Research on Intractable Diseases from the Japanese Ministry of Health, Labor and Welfare. The first author received scholarship support from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
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