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Experimental CELL RESEARCH
Research Article
Different expression of protein kinase A (PKA) regulatory subunits in cortisol-secreting adrenocortical tumors: Relationship with cell proliferation
G. Mantovaniª,1, A.G. Laniaª,1, S. Bondionia, E. Peverellia, C. Pedronia, S. Ferrerob, C. Pellegrinib, L. Vicentinic, G. Arnaldid, S. Bosarib, P. Beck-Peccoza, A. Spadaª,*
ªEndocrine Unit, Department of Medical Sciences, University of Milan, Fondazione Policlinico IRCCS, Milan, Italy
bPathology Unit, Department of Medicine, Surgery and Dentistry, A.O. San Paolo and Fondazione Policlinico IRCCS, University of Milan, Italy “Endocrine Surgery, Fondazione Ospedale Maggiore Policlinico IRCCS, Milan, Italy
ª Azienda Ospedaliero-Universitaria Ospedali Riuniti di Ancona, Italy
ARTICLE INFORMATION
Article Chronology: Received 26 May 2007 Revised version received 29 August 2007
Accepted 29 August 2007 Available online 5 September 2007
Keywords: Adrenocortical tumors PKA
CAMP Proliferation Apoptosis
ABSTRACT
The four regulatory subunits (R1A, R1B, R2A, R2B) of protein kinase A (PKA) are differentially expressed in several cancer cell lines and exert distinct roles in growth control. Mutations of the R1A gene have been found in patients with Carney complex and in a minority of sporadic primary pigmented nodular adrenocortical disease (PPNAD). The aim of this study was to evaluate the expression of PKA regulatory subunits in non-PPNAD adrenocortical tumors causing ACTH-independent Cushing’s syndrome and to test the impact of differential expression of these subunits on cell growth. Immunohistochemistry demonstrated a defective expression of R2B in all cortisol-secreting adenomas (n=16) compared with the normal counterpart, while both R1A and R2A were expressed at high levels in the same tissues. Conversely, carcinomas (n=5) showed high levels of all subunits. Sequencing of R1A and R2B genes revealed a wild type sequence in all tissues. The effect of R1/R2 ratio on proliferation was assessed in mouse adrenocortical Y-1 cells. The R2-selective cAMP analogue 8-Cl-cAMP dose-dependently inhibited Y-1 cell proliferation and induced apoptosis, while the R1-selective cAMP analogue 8-HA-CAMP stimulated cell proliferation. Finally, R2B gene silencing induced up-regulation of R1A protein, associated with an increase in cell proliferation. In conclusion, we propose that a high R1/R2 ratio favors the proliferation of well differentiated and hormone producing adrenocortical cells, while unbalanced expression of these subunits is not required for malignant transformation.
@ 2007 Elsevier Inc. All rights reserved.
Introduction
The pathogenesis of benign and malignant adrenocortical tumors responsible for the ACTH-independent cortisol hyper-
secretion in Cushing’s syndrome is only partially defined. In particular, genetic alterations, such as loss of heterozygosity at 17p1, 2p13 and 11p15 loci and overexpression of insulin-like growth factor-II, cyclin E and more recently B-catenin, have
* Corresponding author. Department of Medical Sciences, Endocrine Unit, Fondazione Ospedale Maggiore Policlinico IRCCS, Via F.Sforza, 35, 20122-Milan, Italy. Fax: +39 02 50320605. E-mail address: anna.spada@unimi.it (A. Spada).
1 Contributed equally to this work and should both be considered first authors.
0014-4827/$ - see front matter @ 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2007.08.024
been associated with malignant adrenocortical lesions, al- though these alterations are not exclusive of carcinomas [1,2]. Based on the notion that proliferation, differentiation and steroid synthesis mostly depend on adrenocorticotropic hormone (ACTH)-induced cAMP generation in the adrenal cortex, it has been proposed that alterations in the cAMP cascade may result in the activation of cortisol synthesis and secretion and the formation of adrenocortical neoplasia. Indeed, previous studies identified in subsets of cortisol- secreting neoplasia either activating mutations of GNAS1 gene, the gene encoding the guanine nucleotide binding protein & stimulating activity polypeptide, or expression of aberrant hormone receptors coupled to Gs protein, such as gastric inhibitory polypeptide (GIP) and luteinizing hormone (LH) receptors, both alterations leading to cAMP pathway activation [3-5]. Recently, genetic defects downstream the cAMP production and involving one of the four genes encoding the PKA regulatory subunits (R1A, R1B, R2A and R2B) have been identified. In particular, germ-line inactivating muta- tions of the gene encoding R1A (PRKAR1A) resulting in increased PKA activity in response to cAMP have been found in patients with Carney complex (CNC), an autosomal dominant multiple neoplasia syndrome that includes as most frequent endocrine feature primary pigmented nodular adrenocortical disease (PPNAD) [6-8]. Subsequently, PRKAR1A mutations have been detected in sporadic non-CNC PPNAD [9] and in a small subgroup of sporadic non-PPNAD adrenocor- tical adenomas [10]. More recently, a genome-wide scan identified mutations in the gene encoding phosphodiesterase 11A4 (PDE11A) in individuals with inherited adrenocortical hyperplasia [11].
Changes of PKA regulatory subunit expression in tumors are not exclusively due to mutational events. Indeed, our group recently demonstrated that in the absence of PRKAR1A mutations tumoral pituitary cells were characterized by low or absent expression of the R1A subunit protein due to protea- some-dependent protein degradation [12]. Moreover, in anal- ogy with the proliferative phenotype resulting from loss of function PRKAR1A mutations in Carney complex, unbalanced expression of PKA R1 and R2 subunits resulted in cAMP- dependent proliferation of somatotroph cells [12]. Although in contrast to previous studies indicating that in a variety of human cell lines transformation coincides with an increase in R1 over R2 expression [13-15], these data strongly suggest distinct roles for these isoenzymes in growth control, depend- ing on the cell system.
The aims of this study were to evaluate the relative expression of the different PKA regulatory subunits in both benign and malignant adrenocortical tumors causing ACTH- independent Cushing’s syndrome and to examine the effect of their selective activation on the proliferation of adrenocortical cells.
Materials and methods
Adrenal tissue samples and cell lines
The study included 21 sporadic adrenocortical tumors, without evidence of associated multiple endocrine neopla-
| Table 1 - Clinical features of secreting adrenal tumors included in the study | ||||||
|---|---|---|---|---|---|---|
| Case | Function state | McFarlane staging | Weiss score | PKA regulatory subunit expression (IHC) | ||
| R1A | R2A | R2B | ||||
| N1 | – | - | - | 3 | 3 | 3 |
| N2 | – | - | - | 3 | 3 | 3 |
| 1 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 2 (AA) | GC | I | 0 | 3 | 3 | 0 |
| 3 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 4 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 5 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 6 (AA) | GC | I | 0 | 3 | 2 | 0 |
| 7 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 8 (AA) | GC | I | 1 | 3 | 3 | 0 |
| 9 (AA) | GC | I | 0 | 3 | 2 | 0 |
| 10 (AA) | GC | I | 1 | 2 | 3 | 0 |
| 11 (AA) | GC | I | 0 | 3 | 3 | 0 |
| 12 (AA) | GC | I | 0 | 3 | 3 | 1 |
| 13 (AA) | GC | I | 1 | 3 | 3 | 0 |
| 14 (AA) | GC | I | 0 | 3 | 3 | 0 |
| 15 (AA) | GC | I | 1 | 2 | 3 | 1 |
| 16 (AA) | GC | I | 0 | 3 | 3 | 0 |
| 17 (AC) | GC | II | 4 | 3 | 3 | 3 |
| 18 (AC) | GC+A | IV | 6 | 3 | 3 | 3 |
| 19 (AC) | GC | IV | 4 | 3 | 3 | 3 |
| 20 (AC) | GC+A | IV | 5 | 3 | 2 | 3 |
| 21 (AC) | GC | IV | 7 | 3 | 3 | 3 |
N, normal adrenal gland removed from 2 patients operated for kidney carcinoma; AA, adrenal adenoma; AC, adrenal carcinoma; GC, glucocorticoids; A, androgens; IHC, immunohistochemistry; PKA R1A, PKA R2A and PKA R2B immunoreactivities grade, 0=absence of immunoreactivity, 1 =< 10%, 2=10-50% and 3⇒50% in at least 400 cells in the main representative high power field.
sia (i.e. Multiple Endocrine Neoplasia type 1, Carney Complex and McCune Albright syndrome). Sixteen were adenomas and 5 were carcinomas, all causing ACTH- independent Cushing’s syndrome, as indicated by high urinary and serum cortisol levels, undetectable ACTH levels and absent cortisol suppression after 8 mg dexamethasone. Clinical features and tumor staging are summarized in Table 1. When possible, normal peri-tumoral adrenal tissue was also collected. Moreover, normal adrenal glands removed from 2 patients operated for kidney carcinoma were used as control. Finally, 9 benign non-secreting adenomas, the so-called adrenal incidentalomas, were also included for immunohistochemistry (IHC), as further con- trol. Small sample fragments were fixed for IHC and the remaining tissues were quickly frozen for subsequent molecular analysis. The transformed mouse adrenal Y-1 cell line was placed in the appropriate sterile culture medium according to the manufacturer’s directions (ATCC; Rockville, MD, USA) for proliferation and apoptosis assays and IHC. In preliminary experiments, cells were incubated with 10 nM ACTH (Sigma-Aldrich, Milan, Italy) for 30 min. at 37 ℃ and intracellular cAMP levels measured in cell extracts, as previously described [16]. Informed consent from patients and the local Ethics Committee approval were obtained.
PK AR1A and PKA R2B sequencing analysis
Genomic DNA was extracted with the phenol-chloroform method from adenomatous tissues (Nucleon-Amersham Life Science, England). The 12 exons and flanking intronic seq- uences of the PRKAR1A gene (GenBank accession no. NM 20002734) as well as the 11 exons and flanking intronic regions of the PRKAR2B gene (GenBank accession no. NM 20002736) were amplified by polymerase chain reaction (PCR) (primers and amplification conditions available on request). Direct sequencing of the amplified fragments was then performed using the AmpliTaq BigDye Terminator kit and 310 Genetic Analyzer (Perkin Elmer Corp., Applied Biosystems, Foster City, CA). Moreover, genetic analysis of the Gsa gene (the so-called gsp oncogene) was performed in all adrenal tumors, as pre- viously described [12].
Immunohistochemistry
Sections from paraffin-embedded tissues from surgically re- moved adrenocortical tumors were processed for IHC, as previously reported [12]. Specific monoclonal antibodies for PKA R1A, R2A and R2B were used under the conditions specified by the manufacturer (BD Transduction Laboratories, Lexington, UK). Antigen-antibody detection was performed using the DAKO ChemMate En Vision detection kit (DAKO A/ S, Glostrup, Denmark) according to the manufacturer’s instructions. Sections were stained with 3,3’-diaminobenzi- dine substrate, counterstained with Meyer hematoxylin and slides prepared for light microscopy examination, as previ- ously reported [12]. Negative controls were obtained by occulting the primary antibody or by using an unrelated mouse monoclonal antibody. At least two blinded readers graded the specimens for all stainings. Briefly, PKA R1A, PKA R2A and PKA R2B immunoreactivities were graded 0-3, with 0=absence of immunoreactivity, 1 =< 10%, 2=10-50% and 3⇒50% in at least 400 cells in the main representative high power fields.
Western blot analyses
Western blot analyses of PKA R1A, R2A and R2B were per- formed in all adrenal samples with the same monoclonal antibodies used for IHC, detected by chemiluminescent method and the resulting bands evaluated by imaging densitometer (BioRad GS-670) [12]. Experiments were repeated at least twice.
mRNA quantitative analysis
Total RNA was isolated from tissue specimens using a com- mercial kit, Trizol (Invitrogen S.R.L., Italy) according to the manufacturer’s instructions and 200 ng RNA was reverse transcribed (Applied Biosystems). PKA R1A, PKA R2A and PKA R2B mRNA levels in adrenocortical tumors were evaluated by real-time quantitative RT-PCR based on Taq- Man methodology, using the ABI Prism 7700 Sequence Detection System (Applied Biosystems). PKA R1A, R2A and R2B mRNA expression were determined applying the 44Ct method, as previously described [12]. To normalize the amount of total RNA added to each reaction mixture,
we quantified as internal RNA control the B-actin (ACTB) mRNA [12].
PKA activity
PKA activity was measured, as previously reported [12], using a non-radioactive PKA kinase activity assay kit (Stressgen, Victoria BC, Canada) in 2 µg of cell extracts from Y-1 cells (transfected with either scrambled or R2B siRNA). The assay is based on a solid phase enzyme-linked immunoabsorbent assay (ELISA) that utilizes a specific synthetic peptide as a substrate for PKA (kempeptide) and a polyclonal antibody that recognizes the phosphorylated form of the substrate. PKA activity reflects the enzymatic activity after stimulation with 5 µM cAMP, free PKA activity represents basal activity, in the absence of cAMP stimulation, and total PKA activity is calculated as the difference between cAMP-stimulated PKA and the PKA inhibited by the protein kinase inhibitor PKI (5 µM).
Cell proliferation
The proliferation of the mouse Y-1 adrenal cells was assessed by colorimetric measurement of 5-bromo-2’-deoxyuridine (BrdU) incorporation during DNA synthesis in proliferating cells (Cell Proliferation Biotrak Elisa, Amersham, Piscataway, NJ), as previously reported [12]. Briefly, cells were cultured in 96-well plate (20,000 cells/well) in the presence of test substances [8-Cl cAMP (8-chloroadenosine-3’,5’-cyclic mono- phosphate), 8-Br cAMP (8-bromoadenosine-3’,5’-cyclic mono- phosphate) and the cell permeant protein kinase A inhibitor PKI from Sigma-Aldrich, Milan, Italy; 8-HA-CAMP (8-n-hexyla- minoadenosine-3’,5’-monophosphate) and 8-PIP-CAMP (8- piperidinoadenosine-3’,5’-cyclic monophosphate) from Bio- log, Germany] for 96 h at 37 ℃ and then with BrdU for 2 h to allow BrdU incorporation in newly synthesized cellular DNA. Proliferation was expressed as relative fluorescence units (RFU). All experiments were repeated at least 3 times on 2 different clones, and each determination was done in quintuple.
Evaluation of apoptosis
Apoptosis was assessed by caspase-3 activity determination. Caspase-3 enzymatic activity was measured using Apo-ONE™ Homogenous Caspase-3 Assay (Promega Corp.), a fluorescent assay based on cleavage of the non-fluorescent caspase substrate Z-DEVD-R110 by caspase-3 to create the fluorescent Rhodamine 110, as previously described [17]. Cells were seeded in 96-well plates (50,000 cells/well) and treated with different agents for 24 h at 37 ℃, in DMEM supplemented with 10% FCS. Fluorescence was measured at an excitation at 485± 20 nm and an emission at 530±25 nm. Caspase-3 activity was indicated by net fluorescence (assay RFLU- blank RFLU). Experiments were repeated at least twice and each determi- nation was done in triplicate.
Synthesis and transfection of siRNA
Small interfering RNA (siRNA) for mouse R2B gene was synthesized by Dharmacon (Chicago, IL, USA), and Y-1 cell line was transfected with the double stranded RNA through
A. Carcinoma
Adenoma
C.
NAAAACC
R1A
R1A
R2A
R2A
R2B
GAPDH
R2B
D.
* p<0.01
☒ Normal adrenal
☐ Adrenal adenoma
200
B.
Protein expression (%)
☐ Adrenal carcinoma
150
N
100
R2B
50
*
A
0
Adenoma
R1A
R2A
R2B
JetsiENDO transfection Reagent (Polyplus transfection), according to manufacturer’s instructions. Y-1 cells were exposed to double-stranded RNA and transfection reagent for 96 h before performing proliferation assay. Western blot analysis was carried out with the specific R2B and R1A antibodies in order to control the effective silencing of the R2B gene and the subsequent R1A up-regulation, respectively. Corresponding scrambled siRNA for the same regulatory subunit and siRNA for GAPDH were used as internal negative and positive controls, respectively, as previously reported [12]. Each experiment was repeated at least twice.
Statistical analysis
The results are expressed as the mean+SD. A paired or un- paired two-tailed Student’s t test was used to detect the sig- nificance between two series of data. P<0.05 was accepted as statistically significant.
Results
PKA R1A and PKA R2B sequencing analysis
Analysis of the 12 exons and flanking regions of PRKAR1A and the 11 exons and flanking regions of PRKAR2B did not reveal
mutations of the genes in none of the tumors included in the study. Two known polymorphisms [18] in the non-coding sequence of PRKAR1A, i.e. a T insertion in intron 3 (exon 4 IVS- 5) and a base substitution (A to C) in the 5’-untranslated region of exon 1A, were found in 2 and 3 tumors, respectively. A known polymorphism in the coding region of PRKAR2B (a C to G substitution with no change in the amino acid sequence in exon 11) was detected in 5 tissues [19].
No activating mutations of the Gsa gene were detected in any of the tumors included in this study.
PKA regulatory subunit expression
At the protein level, R2B subunit was absent at immunohisto- chemistry in 9 functioning adenomas and barely detected in the remaining 7, while R1A and R2A were strongly expressed in all adenomas (Table 1, Figs. 1A and B). The loss of R2B was restricted to the tumoral tissue since the normal surrounding tissue was strongly positive for all the R subunits, as were normal tissues from patients operated for kidney carcinoma (Table 1, Fig. 1B). Similarly, adrenocortical carcinomas showed immunoreactivity for all the 3 regulatory subunits of PKA (Table 1, Fig. 1A). In contrast with cortisol-secreting adenomas and similarly to adrenal cancers and normal adrenal samples, benign non-secreting adenomas displayed a strong immuno- reactivity for the R2B antibody. R1B was not considered as its
expression has been typically associated to the central nervous system and to date it has never been described in peripheral tissues. The data obtained by immunohistochemistry were confirmed in all the samples included in the study by evaluating R1A and R2B protein levels by western blot analysis with subsequent quantification of the resulting bands (Figs. 1C and D). Finally, real-time PCR of PKA R1A, R2A and R2B was performed in normal adrenal cortex and in all adrenal tumors were included in the study. Quantitative analysis of the different PKA regulatory subunits showed detectable, although variable, mRNA levels in all adenomas and carcinomas, with no significant difference between the two entities (data not shown).
Effect of cAMP analogues on cell proliferation and apoptosis
We investigated the role of the different PKA regulatory subunits on the proliferation of Y-1 cells that are characterized
A.
8 HA-CAMP
RIA
8 CI-CAMP
R2B
Cell proliferation (% modification)
A 8 Br-cAMP
Bas 8-HA 8 CI
150
* P < 0.01
*
100
*
50
0
*
-50
*
-100
5 µM
10 AM
100 μΜ
B.
0,6
*
Cell proliferation (RFU)
0,5
1
0,4
0,3
cos
0,2
T
0,1
*
T
0
Basal
8 CI
8 CI
8-HA 10μ.Μ
8-HA 10μΜ
100 µM
100μΜ
+PKI
+PKI
C.
20000
*
Caspase 3 activity (RFU)
19000
cos
18000
17000
16000
Basal
8 CI
8 CI
8 Br
8 C1
10 μΜ
100 μΜ
100 μΜ
100μΜ
+PKI
by a strong positivity for all the 3 PKA subunits and therefore easy to handle to this purpose. In these cells, ACTH (10 nM) caused a 2.5-fold increase in intracellular cAMP levels, demonstrating the integrity of the receptor-effector coupling. The addition of 8-Cl cAMP, a cAMP analogue able to selectively activate R2 subunits [13], caused a significant dose-dependent inhibition of cell proliferation (Figs. 2A and B) that was detectable at concentrations higher than 5 uM and maximal at 100 µM (% inhibition: 82±12% vs. basal levels; P=0.0005). Co- incubation with the PKA inhibitor PKI (5 M) resulted in a significant reduction of this effect (% inhibition: 33±8% vs. basal levels) (Fig. 2B). The effects obtained with R2 activation were accompanied by a marked increase in R2B levels together with a reduction in R1A levels, as assessed by western blot analysis (Fig. 2A, upper panel), suggesting that the observed anti-proliferative effect was indeed determined by R1A abolishment and R2B induction.
Cell treatment with 8-HA-CAMP, in association with 8-PIP- CAMP, induced a dose-dependent increase in cell proliferation (maximum effect: 50±15% at 10 µM, P=0.002; Figs. 2A and B) that was partially reverted by the addition of PKA inhibitor PKI. 8-PIP-CAMP is a site selective cyclic AMP analogue with high selectivity for site A of PKA type 1 and for site B of type 2, acting
Fig. 2 - Effects of different cAMP analogues on Y-1 cultured cell proliferation and apoptosis. (A) 8-Cl-cAMP, a cAMP analogue able to selectively activate R2 subunit, caused a significant and dose-dependent inhibition of Y-1 cell proliferation. Conversely, a significant increase in Y-1 cell proliferation was observed after selective R1A activation by incubation with 8-HA-CAMP in association with 8-PIP-CAMP. The same was not true with the non-selective PKA activator 8-Br-cAMP. The effects obtained with R2 activation were accompanied by a marked increase in R2B levels together with a reduction in R1A levels, as assessed by western blot analysis showed in the upper panel, suggesting that the observed anti-proliferative effect was indeed determined by R1A abolishment and R2B induction. Data are expressed as percentage vs. non-stimulated cells and represent the mean of determinations carried out in quintuple in at least three independent experiments. (B) The effects induced by 8-Cl-cAMP and 8-HA-CAMP were partially abolished by PKA blockade with PKI. Data are expressed as Relative Fluorescence Units (RFU) and are the mean ± SD of cell number determinations carried out in quintuple in at least three independent experiments. * P<0.01 vs. basal, §P<0.05 vs. 8-Cl-CAMP 100 L.M; ^P <0.05 vs. 8-HA-CAMP 10 LM+8-PIP-CAMP 90 p.M. (C) Effect of different 8-Cl-cAMP concentrations on apoptosis in cultured Y1 cells. A significant increase in caspase-3 activity was observed after 8-Cl cAMP incubation (100 µM), this effect being partially abolished by PKA blockade with PKI. No effect on caspase-3 activity was observed after incubation with 8-Br CAMP (100 µM). Caspase-3 activity was indicated by net fluorescence (assay RFLU- blank RFLU). Experiments were repeated at least twice and each determination was done in quintuple. Data represent the percent increase (mean±SD) of caspase-3 activity over basal values. * P<0.01 vs. basal; $P <0.01 vs. 8-Cl-CAMP.
A.
B. 0,4
* P<0.05 vs control
*
T
Cell proliferation (RFU)
+ RIA
0,3
T
T
R2B
0,2
Basal C siR2B
0,1
0
Basal
Control
siR2B
C.
0,4
& *
-
Basal
PKA activity (RFU)
*
2 CAMP
&
CAMP + PKT
-
0,2
4
con
* P<0.05 vs basal
a
4
& P<0.05 vs control
0
Control
siR2B
synergistically with other analogues having opposite site- selectivity. In particular, combination of 8-PIP-cAMP with 8- HA-CAMP, a site-selective analogue which prefers site B of PKA 1, allows preferential activation of PKA type 1 [20]. The stimulatory effects of 8-HA-CAMP were partially mimicked by the non-selective activation of both R1 and R2 by 8-Br-cAMP [13] that caused a reproducible but not significant increase in cell proliferation (15±5% over basal levels) at 100 µM (Fig. 2A).
Finally, the exposure of cultured Y-1 cells to 8-Cl cAMP but not to 8-Br cAMP induced a dose-response increase in caspase- 3 activity, that reached statistic significance at 100 uM (28±4% vs. basal; P=0.007) and was almost totally abrogated by the PKA inhibitor PKI (Fig. 2C).
R2B gene silencing
The effect of changes in R1A/R2B ratio on cell proliferation was further investigated in Y-1 cells by silencing R2B RNA expression by siRNA transfection. Immunocytochemical anal- ysis of transfected cells showed that the R2B expression loss was actually always limited to a subset of cells, indicating the lack of optimized transfection efficiency. In particular, the maximal reduction of R2B protein levels obtained by this manipulation (48% vs. basal) was associated with the up- regulation of R1A protein and a significant induction of Y-1 cell proliferation (26±5% vs. control, P=0.02; control=cells transfected with scrambled R2B siRNA) (Figs. 3A and B). Accordingly, this manipulation was accompanied by a mod-
est, yet significant increase in both free and cAMP-stimulated PKA activity (16±4% and 18±40.7% vs. control, respectively; P=0.046 and 0.036) (Fig. 3C). Experiments were repeated at least twice and each determination was done in triplicate.
Discussion
The present study provides evidence for a novel alteration in the cAMP-dependent pathway, i.e. the defective expression of the PKA regulatory subunit 2B (R2B) protein, in adrenocortical adenomas causing ACTH-independent Cushing’s syndrome. In fact, the normal adrenal cortex showed a great abundance of both R1 and R2 subunits, that were not influenced by ACTH secretion, being similar in conditions characterized by sup- pressed (i.e. Cushing’s syndrome) and normal (i.e. normal adrenal gland removed from 2 patients operated for kidney carcinoma and benign non-secreting adenomas) ACTH levels. Interestingly, the defective expression of R2B only occurred in benign, hormone secreting tumors whereas secreting carci- nomas and non-secreting adenomas showed high levels of both R1 and R2.
Consistent with the absence of mutations in the coding regions of PRKAR2B gene, R2B mRNA was detected in all adrenocortical tumors, independently from the benign or ma- lignant phenotype as demonstrated by real-time PCR experi- ments. The discrepancy between R2B mRNA and protein levels found in adrenocortical adenomas suggests the exis- tence of a high rate of R2B protein degradation and/or alte- rations in protein targeting in these tissues. Taking into account that within the cell R subunits, and particularly R2, are tightly bound to scaffold molecules, such as the A-kinase anchoring proteins (AKAPs), that are required for compart- mentalization, targeting and signaling, it is tempting to speculate that the defective expression of R2B at the protein level may be related to altered coupling to AKAPs [21].
All adrenocortical adenomas displayed a strong preva- lence of R1 over R2 protein, since the low or absent R2B was associated with high R1A levels. Therefore, we asked which was the impact of the high R1/R2 ratio on the proliferation of cells of the adrenocorticotroph lineage, taking into account that PKA activation may exert different and even opposite actions on cell growth according to the cell type [22]. Since in preliminary experiments primary cultures from adrenal tumors showed a limited life span and were contaminated by the presence of non-steroidogenic cells (unpublished observations), we used adrenal cell lines to investigate the impact of R1/R2 ratio on cell growth. In particular, in the present study, the manipulation of R1 and R2 expression by long-term exposure to selective agonist and by siRNA transfection was carried out on Y-1 adrenocortical cells, a murine cell line derived from a minimally deviated tumor that maintained the differentiated phenotype [23]. This cell line has been extensively used as a model system for research on adrenocortical function. In particular, the clones included in this study were characterized for the presence of a normal receptor-effector coupling, as indicated by the ACTH-induced cAMP stimulation and, most importantly for the aim of the present study, by high levels of both R1 and R2 subunits.
The data obtained showed an important impact of R1/R2 ratio on adrenocortical cell growth. First, the selective activation of R2B and the consequent decrease of the R1/R2 ratio induced by 8-Cl-cAMP caused a significant inhibition of Y-1 cell proliferation. Second, in agreement with the results obtained in other cell lines [24], this agent was effective in inducing apoptosis, as indicated by the increased enzymatic activity of caspase-3, a major protease mediator of the extrinsic and intrinsic apoptotic pathways [25]. While the proapoptotic action of 8-Cl-cAMP was mostly cAMP-dependent, the anti- proliferative response was only partially abrogated by specific PKA inhibitors, suggesting the involvement of PKA-dependent and independent pathways in this effect. Indeed, the interac- tions of the regulatory and catalytic PKA subunits with other signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, have been documented in several cell lines, including Y-1 cells [26]. Third, R1A selective agonists caused a significant stimulation of Y-1 proliferation, further supporting the involvement of R1/R2 ratio decrease in the inhibitory signal elicited by 8-Cl-cAMP. Finally, increased cell growth was also observed after silencing R2B expression by siRNA transfection, a manipulation that mimicked the expres- sion pattern found in adrenocortical adenomas.
Taken together, these data indicate that the induction of a high R1/R2 ratio resulted in adrenocortical cell proliferation whereas the activation of the R2 subunit resulted in cell growth inhibition and apoptosis. These data are consistent with previous studies supporting the view that R1 was related to cell proliferation whereas R2 was primarily involved in differentiation [13-15]. Accordingly, in a variety of cell systems transformation coincide with a sharp increase in R1, while R2 overexpression reverts the tumoral phenotype [27,28]. As far as adrenocortical cells are concerned, mutants from Y-1 cells harboring dominant-negative mutations in the R1A gene have been used to establish obligatory roles for cAMP and PKA in ACTH-regulated steroidogenesis and in cell growth stimula- tion and inhibition [26,29]. Indeed, the role played by ACTH in the control of cell proliferation is still controversial, although most data seem to indicate anti-mitogenic effects of this agent in adrenal cells [26,30,31]. It will be of interest to further evaluate whether the variable and even opposite cAMP- dependent actions of ACTH might be associated with differ- ential induction of R1 and R2 subunits expression.
The present data indicating that a high R1/R2 ratio represents an alteration favoring adrenocortical cell growth seem to be in striking contrast with the defective expression of R1A due to PRKAR1A mutations that has been found in PPNAD associated or not with Carney complex and in a small subgroup of sporadic non-PPNAD adrenocortical adenomas [5-9]. However, considering that defective R1A facilitates the dissociation and release of the two PKA catalytic subunits that in turn phosphorylate a variety of substrate proteins [5-7], it is conceivable that the absent or low expression of R2B might induce a similar phenotype in the adrenocortical cell. Indeed, although some morphological features, such as massive deposition of lipofuscin pigment and small size of the nodular lesions, and some biochemical characteristics, such as para- doxical cortisol responses to dexamethasone, are frequently present in PPNAD and absent in adrenocortical adenomas causing ACTH-independent Cushing’s syndrome, the defi-
ciencies of R1A and R2B are both associated with adrenocor- tical tumors characterized by hormone overproduction and benign nature. Therefore, the present data, although appar- ently contradictory with the previously reported loss of R1A, support the view that activation of the cAMP-dependent pathway induced either by genetic abnormalities, such as gsp oncogene, PRKAR1A and phosphodiesterase 11A4 (PDE11A) loss of function mutations and aberrant expression of Gs-coupled receptors, or by the reduced expression of R2B protein here reported, is associated with the proliferation of well differentiated cortisol-secreting adrenocortical cells.
In contrast to the defective R2B expression observed in secreting adenomas, no modification in R1/R2 ratio was present in cortisol-secreting carcinomas and benign non- secreting adenomas, once again indicating that low or absent R2 expression may underlay cAMP-dependent proliferation exclusively of well differentiated hormone producing cells. Moreover, this observation further supports a minor if any role of cAMP-dependent PKA signaling pathway in adrenocortical malignant transformation. This conclusion is consistent with the absence of activating GNAS1 mutations and aberrant Gs coupled receptors as well as the low expression of cAMP- responsive element binding protein (CREB) found in adreno- cortical carcinomas [32]. Moreover, these in vitro observations are in agreement with clinical experiences reporting no cases of adrenal cancer in PPNAD patients. Admittedly, since the study was carried out on differentiated adrenocortical carcinomas, as indicated by hormone hypersecretion, the presence of alterations in less differentiated cancers cannot be ruled out.
In conclusion, the present study demonstrates that defi- ciency of the PKA regulatory subunit R2B is a common event in non-PPNAD adrenocortical adenomas causing Cushing’s syn- drome and provides evidence for a proliferative role of the unbalanced expression of PKA isoenzymes in adrenocortical cells. Although the molecular events representing the first “hit” for the formation of adenocortical adenomas, in the majority monoclonal, are still unknown [1-3,33], in this study it is proposed that a high R1/R2 ratio may create the environment necessary for developing this neoplastic phenotype. By contrast, changes in R subunit expression are not required for the malignant transformation of adrenocortical cells.
Acknowledgments
This work was partially supported by AIRC (Associazione Italiana Ricerca Cancro, Milan), PRIN grant 2006060982_002 and Ricerca Corrente Funds of Fondazione Policlinico IRCCS (Milan).
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