Endocrinology
Inactivation of the Carney Complex Gene 1 (Protein Kinase A Regulatory Subunit 1A) Inhibits SMAD3 Expression and TGF3-Stimulated Apoptosis in Adrenocortical Cells
Bruno Ragazzon,12 Laure Cazabat,12 Marthe Rizk-Rabin,1,2 Guillaume Assie,1,2,3 Lionel Groussin,1,2, Hélène Fierrard,1,2 Karine Perlemoine,1,2 Antoine Martinez,4 and Jérôme Bertherat1,2,3
“Institut Cochin, Université Paris Descartes, Centre National de la Recherche Scientifique (UMR 8104); 2Institut National de la Santé et de la Recherche Médicale, U567; 3Assistance Publique Hôpitaux de Paris, Hôpital Cochin, Department of Endocrinology, Reference Center for Rare Adrenal Diseases, Paris, France and ‘Centre National de la Recherche Scientifique UMR6247, Génétique Reproduction et Développement, Clermont Université, Aubière, France
Abstract
The cyclic AMP signaling pathway can be altered at multiple levels in endocrine tumors. Its central component is the protein kinase A (PKA). Carney complex (CNC) is a hereditary multiple neoplasia syndrome resulting from inactivating mutations of the gene encoding the PKA type I & regulatory subunit (PRKAR1A). Primary pigmented nodular adrenocorti- cal disease is the most frequent endocrine tumor of CNC. Transforming growth factor 3 (TGF3) regulates adrenal cortex physiology and signals through SMAD2/3. We used an interference approach to test the effects of PRKAR1A inactivation on PKA and TGFß pathways and on apoptosis in adrenocortical cells. PRKAR1A silencing stimulates PKA activity and increases transcriptional activity of a PKA reporter construct and expression of the endogenous PKA target, NR4A2, under basal conditions or after forskolin stimulation. PRKAR1A inactivation also decreased SMAD3 mRNA and protein levels via PKA, altering the cellular response to TGF3. SMAD3 expression was also inhibited by adrenocorticorticotropic hormone in the mouse adrenal gland and by forskolin in H295R cells. TGFß stimulates apoptosis in H295R cells, and this effect was counteracted by PRKAR1A inactivation. PRKAR1A silencing decreased the percentage of apoptotic cells and the cleavage of apoptosis mediators [caspase-3, poly(ADP-ribose) polymerase, and lamin A/C]. Inactivating mutations of PRKAR1A observed in adrenocorti- cal tumors alter SMAD3, leading to resistance to TGFß- induced apoptosis. This cross-talk between the PKA and the TGF3 signaling pathways reveals a new mechanism of endocrine tumorigenesis. [Cancer Res 2009;69(18):7278-84]
Introduction
The cyclic AMP (cAMP) pathway plays a central role in cellular differentiation and proliferation. Activation of this pathway often inhibits cellular proliferation; by contrast, it can stimulate cell proliferation in several endocrine tissues (1, 2). Indeed, molecular defects in the cAMP/protein kinase A (PKA) pathway occur in endocrine tumors. These key components include G protein-coupled
seven-transmembrane receptors, stimulatory G protein subunit, phosphodiesterase, and PKA (3-7). In most cases, the tumors harboring these genetic defects are well differentiated, oversecreting endocrine tumors. In vivo approaches to mimic these genetic alterations can induce hyperplasia and/or tumor formation (8-12). However, the mechanisms leading to cellular dysregulation by molecular alterations of the cAMP/PKA pathway are still poorly understood.
PKA is a heterotetramer made of two regulatory subunits and two catalytic subunits. Regulatory subunits are encoded by four different genes (PRKAR1A, PRKAR2A, PRKAR1B, and PRKAR2B), and three genes encode the catalytic subunits (PRKACA, PRKACB, and PRKACG). The catalytic subunits dissociate after two cAMP molecules bind to the regulatory subunits. Germ-line inactivating mutations of PRKAR1A are responsible for the syndrome of cardiac myxomas, spotty skin pigmentation, and endocrine overactivity or Carney complex (CNC; refs. 7, 13, 14). Growth hormone-secreting pituitary adenomas, thyroid tumors, testicular tumors, and primary pigmented nodular adrenocortical disease (PPNAD), which is responsible for Cushing’s syndrome, are observed in CNC. PPNAD is the most frequent endocrine manifestation of CNC, and germ-line PRKAR1A inactivating mutations occur in patients with isolated PPNAD (15, 16). Somatic mutations of PRKAR1A also occur in secreting adrenocortical adenomas (17). Most mutations lead to a premature stop codon that gives rise to an unstable mRNA degraded by nonsense-mediated mRNA decay (NMD). PKA activity is higher in tumors with PRKAR1A inactivating mutations (7). Moreover, a PKA interaction with the mitogen-activated protein kinase pathway has been observed in primary cell culture of PPNAD tissues (18).
Transforming growth factor ß (TGFB) represents a large family of growth and differentiation factors that include activins, inhibins, and bone morphogenetic proteins. TGFß regulates proliferation, differentiation, adhesion, cell migration, angiogenesis, and apopto- sis. TGFß signals are conveyed through type I and II serine/ threonine kinase receptors to specific intracellular mediators known as SMAD proteins. Vertebrates possess nine SMAD proteins distributed into three classes: class 1, receptor-activated SMADs (SMAD1, SMAD2, SMAD3, SMAD5, and SMAD8); class 2, come- diator SMADs (SMAD4 and SMAD10); and class 3, inhibitory SMADs (SMAD6 and SMAD7; ref. 19).
Activation of the TGFß receptors by ligand binding results in the phosphorylation of type I receptors by the serine/threonine kinase activity of the type II receptor. SMAD2 and SMAD3 act as direct substrates of activated type I receptors. They are phosphorylated on the last two serines at the COOH terminus. Once phosphory- lated, receptor-activated SMADs associate with the comediator SMADs, SMAD4, and the heteromeric complex translocates into
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Requests for reprints: Jérôme Bertherat, Service des Maladies Endocriniennes et Métaboliques, Hôpital Cochin, 27, rue du Faubourg Saint-Jacques, 75014 Paris, France. Phone: 33-1-58-41-18-95; Fax: 33-1-46-33-80-60; E-mail: jerome.bertherat@cch.ap-hop- paris.fr.
@2009 American Association for Cancer Research.
doi:10.1158/0008-5472.CAN-09-1601
A
HEK293
H295R
ATC1
siCtr
SiR1A
siCtr
SiR1A
siCtr
SiR1A
R1A
ß-actin
R1A/ B-actin
1.
0.5
0
siCtr
SİR1A
siCtr
siR1A
siCtr
SİR1A
B
HEK293 siCtr siR1A
siCtr
siR1A
R1A
Protein level
1
R2A
R2B
PKA
0.5
C
ß-actin
0
R2A
R2B
C
the nucleus to activate specific genes (19). TGFß regulates adrenal cortex physiology (20) and is produced by, and interacts with, specific receptors on adrenal cortical cells (21), suggesting that it acts as an intra-adrenal autocrine/paracrine factor. TGFß is also
involved in the regulation of human fetal adrenal development. TGFß may exert an antimitotic effect on human fetal adrenal cells in vitro (22, 23). Moreover, previous studies suggested an interaction between cAMP/PKA and TGFß pathways (24, 25).
Here, we tested the effects of PRKAR1A inactivation on PKA and TGFß pathways and on TGFß-induced apoptosis, particularly in adrenocortical cells.
Materials and Methods
Culture cell lines. Human embryonic kidney cells HEK293, human adrenocortical carcinoma cells H295R, and murine adrenocortical cells ATC1 were cultured as previously described (26, 27). HEK293 cells are used for efficient transfection and to control the siR1A efficiency. Cells were cultured either in six-well plates (500 × 103 per well) for protein extractions or in 12-well plates (300 × 103 per well) for luciferase reporter assays, RNA extraction, and apoptosis assays. Forskolin, adrenocorticorticotropic hormone (ACTH; fragment 1-24), H89, and human TGFß1 are used as indicated in figure legends.
Cell transfection and electroporation. HEK293 cells were transfected with Lipofectamine 2000 reagent (Invitrogen Life Technologies) following the manufacturer’s instructions. H295R and ATCI were electroporated with Nucleofector II (program T20 for H295R and program T30 for ATC1; Amaxa Biosystems). Five million cells were suspended in 100 uL of Amaxa electroporation buffer (kit R). Small interfering RNA (siRNA) used for targeting PRKARIA is UGAAUGGGCAACCAGUGUUdTdT (siRIA), and the siRNA control is CAGUCGCGUUUGCGACUGGdTdT (siCtr; Dharmacon). HEK293 cells were transfected with 80 pmol of siRNA/mL per well, and H295R or ATCI cells were electroporated with 80 pmol of siRNA/1 x 106
A
HEK293
B
HEK293 Reporter constructs
PKA activity
*
2.5
pSom-Luc
siCtr
*
*
siCtr
Relative activity
2
*
siR1A
Relative activity
6-
siR1A
1.5
ns
4
*
1
0.5
2
0
FK
FK
0
FK
FK
PKI
H89
C
HEK293
siCtr
SİR1A
H295R
PRKAR1A
PRKAR1A
1.5
*
1
1
mRNA level
0.5
0.5
0
0
1
3
6
h FK
0
0
1
3
6
h FK
12
NR4A2
NR4A2
9
20
6
10
3
ns
*
ns
ns
ns
0
0
1
3
6
h FK
0
0
1
3
6
h FK
A
HEK293
H295R
siCtr
SiR1A
siCtr
SİR1A
R1A
SMAD3
R1A
SMAD4
SMAD3
ß-actin
ß-actin
SMAD3
SMAD3
Protein level
1
I
Protein level
1
T
*
0.5
T
0.5
*
0
siCtr
SİR1A
0
siCtr
siR1A
B
HEK293 SMAD3
H295R
D
H295R
mRNA level
1-
SERPINE1 (PAI)
*
15
*
0.5
mRNA level
10
0
siCtr siR1A
siCtr siR1A
5
C
ns
HEK293
0
(CAGA)9-Luc
siCtr siR1A
*
iCtr siR1A
Relative activity
120
Control
TGF₿
Protein
siCtr siR1A siCtr siR1A
60
SERPINE1
ß-actin
ns
0
siCtr siR1A
siCtr siR1A
TGF฿
cells. A cAMP/PKA pathway reporter construct driving expression of luciferase gene was used: pSom-Luc (28) with the somatostatin promoter containing a cAMP-responsive element (CRE). (CAGA)9-Luc, an artificial SMAD3/SMAD4-specific recognition sequence, CAGA, cloned upstream of a SV40 minimal promoter and driving luciferase expression (29) was used to study TGFß-regulated transcription. Rous sarcoma virus (RSV)-Renilla that contained the RSV promoter-enhancer inserted upstream of the coding sequence of the Renilla luciferase (Promega Corp.) was used as a control of transfection efficiencies. Cells were cotransfected with 80 pmol of siRNA, 10 ng of the RSV-Renilla, and 250 ng of each luciferase reporter constructs and lysed, and both firefly and Renilla luciferase activities were sequentially measured with the Dual Luciferase Reporter Assay System (Promega). Results are expressed as firefly luciferase activity normalized to Renilla luciferase activity of the same sample. For apoptotic studies, H295R cells were cotransfected with 80 pmol of siRNA and 250 ng of empty pCDNA3+ or SMAD3-Flag vector/1 × 106 cells.
Analysis of RNA by quantitative PCR. The total RNA extracted from cell lines was treated with DNase and further purified with the RNeasy Mini kit and RNase-free DNase Set (Qiagen) according to the manufacturer’s instructions. For mice adrenals, total RNA was isolated with Trizol (Invitrogen Life Technologies) according to the manufacturer’s instructions. Purified RNA was reverse transcribed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and expression levels of target genes were analyzed by quantitative PCR using a LightCycler Fast Start SYBR Green kit (Roche Diagnostics) according to the manufacturer’s instructions. The PCR conditions for all target genes were described in Supplementary Table S1. Relative quantification of target cDNA was determined by calculating the difference in cross-threshold (CT) values after normalization to PPIA (CYCLO) signals (AACT method).
Western blot analysis. Whole-cell or tissue lysates and Western blotting were performed as previously described (27). Antibodies are described in Supplementary Table S2.
PKA assay. The PepTag Non-Radioactive Protein Kinase Assay kit (Promega) was used to measure the activity of PKA, as previously described (30).
Animals and treatments. Animal studies were conducted in agreement with standards described by NIH Guide for Care and Use of Laboratory Animals as well as with the local laws and regulations applicable to animal manipulations in France. Animal studies were conducted on adult male CD1 mice as previously described (27).
Annexin V-FITC staining of apoptotic cells. Apoptotic cell distribution was analyzed by flow cytometry using a FACScan (Epics XL Coulter) as previously described (30).
Statistical analyses. All data with statistical analyses represent the quantification of three experiments ± SE. Control conditions were set as one and data were analyzed using a Mann-Whitney test. Significance was set at P < 0.05 (represented by * in figures).
Results
Efficient inactivation of PRKAR1A by siRNA. RNA interference was used to study the effects of PRKAR1A mutations that lead to mRNA degradation by NMD. PRKAR1A siRNA (siR1A) decreased PRKAR1A protein (R1A) in HEK293 cells as well as the adrenocortical cells H295R and ATC1 (Fig. 1A). Compared with control siRNA (siCtr), the level of RIA protein was 1 ± 0.11 versus 0.14 ± 0.04 (-86%; P < 0.05) in HEK293, 1 ± 0.02 versus
0.45 ± 0.02 (-55%; P < 0.05) in H295R, and 1 ± 0.04 versus 0.73 ± 0.03 (-27%; P < 0.05) in ATC1. Other PKA subunits (R2A, R2B, and C) were not altered by siR1A in HEK293 (Fig. 1B), H295R, or ATC1 cells (data not shown). Differences in siR1A efficien- cy across the cell lines probably reflect different transfection/ electroporation efficiency.
PRKAR1A silencing increases PKA activity and PKA dependent transcription. We then examined the effects of PRKAR1A inactivation on PKA activity and PKA-dependent transcription by (a) an enzymatic activity assay using a PKA synthetic substrate in vitro, (b) reporter gene activity using a PKA- dependent luciferase reporter construct, and (c) gene expression by studying the expression of an endogenous PKA-responsive gene.
a. In HEK293 cells transfected with siR1A, treatment with forskolin for 10 minutes enhanced PKA activity more than siCtr- transfected cells: 2 ± 0.04 siRIA versus 1.7 ± 0.05 siCtr (P < 0.05; Fig. 2A). Similar results were observed in H295R cells, and these effects could be blocked by the specific PKA inhibitor (PKI; Fig. 2A).
b. We used a luciferase reporter driven by the somatostatin promoter containing a cAMP-responsive element (CRE; pSom- Luc). Transfected HEK293 cells showed higher transcriptional activity of the reporter construct both basally and after 6 hours of forskolin stimulation (Fig. 2B). This effect was partly blocked by pretreatment with the PKA inhibitor H89 (Fig. 2B), indicating that the effect of PRKAR1A inactivation on reporter gene activity is due to increased PKA activity. Similar results were observed in the adrenocortical cell lines with pSom-Luc (data not shown)
A
H295R
1
SMAD3
*
Ctrl
H89
*
Protein
mRNA level
+ | - + FK (24 h)
SMAD3
0.5
GAPDH
0
0
1 3 6 9h FK
B
ATC1
0 3 6 12 h ACTH
Protein
StAR
SMAD3
GAPDH
C
In vivo dex 5d
smad3
mRNA level
1-
C 2h 17 h ACTH
*
*
Protein
StAR
0.5
SMAD3
GAPDH
0
C 2h 17h ACTH
dex 5 d
and with another cAMP/PKA pathway reporter construct with a basic promoter element (TATA box) joined to four CRE repeats (data not shown).
c. We measured mRNA levels of a gene target of the ATF/cAMP- responsive element binding protein (CREB) transcription factors-the orphan nuclear receptor NR4A2 (NURR1; Fig. 2C; ref. 31). Forskolin stimulation increased NR4A2 mRNA in HEK293 and H295R cells. The maximal level of NR4A2 mRNA was observed after 1 hour of forskolin treatment in the two cell lines, with forskolin stimulation higher when PRKAR1A was inactivated. As for proteins (Fig. 1A), changes in PRKAR1A mRNA levels were larger in HEK293 than in H295R cells.
PRKARIA silencing and PKA pathway alter TGFß pathway. Because the TGFß pathway plays a role during tumorigenesis in several organs and is known to inhibit steroidogenesis in adrenocortical cells, we measured the effects of PRKAR1A inactivation on this signaling pathway. PRKAR1A silencing decreased SMAD3 levels both at the protein (Fig. 3A) and mRNA levels (Fig. 3B) in HEK293 and H295R cells. SMAD2 mRNA also decreased (data not shown). SMAD4, which is required for SMAD2/ 3 to form a functional heterocomplex for translocation to the nucleus, was not decreased after PKRAR1A inactivation (Fig. 3A).
As SMAD3 is an essential cytoplasmic mediator of the TGFB signaling pathway, we also tested the effect of PRKAR1A silencing on a TGFß-dependent response element using an artificial SMAD3/ SMAD4 reporter plasmid, (CAGA),-Luc. siRIA caused HEK293 cells to produce a lower response from this reporter construct after 24 hours of TGFß stimulation (92 ± 6-fold) than siCtr (121 ± 11-fold; P < 0.05; Fig. 3C). Similarly, in H295R cells, siR1A reduced the induc- tion level of the endogenous TGFß target gene SERPINE1 (PAI) from 13.42 + 1.49-fold with siCtr to 7.73 ± 0.69-fold (P < 0.05; Fig. 3D).
To investigate if this TGFß alteration was due to PKA activity, we tested the effect of forskolin and ACTH on SMAD3 expression in adrenocortical cells. Forskolin stimulation decreased SMAD3 mRNA and protein expression in H295R (Fig. 4A), and ACTH decreased SMAD3 protein in mouse ATC1 cells (Fig. 4B). H89, an inhibitor of PKA, blocked SMAD3 inhibition by forskolin (Fig. 4A), suggesting a PKA-dependent effect.
We next asked whether the forskolin- and ACTH-dependent regulation of SMAD3 expression observed in the adrenocortical cell lines might also occur in vivo. Therefore, we measured changes in the expression of SMAD3 in the adrenal glands of mice injected for 5 days with dexamethasone to maintain a negative feedback on the hypothalamicuitary-adrenal axis in combination with ACTH for increasing periods of time. ACTH increased StAR protein levels (a control) and inhibited SMAD3 proteins (Fig. 4C, left) and smad3 mRNA [0.57 ± 0.12-fold (-43%) after 17 hours versus 1 ± 0.13-fold for control; P < 0.05; Fig. 4C, right].
PRKAR1A silencing protects H295R cells from induced apoptosis. PRKAR1A inactivation in H295R cells modulates cell cycle by increasing slightly the G1 phase and decreasing the G2 phase (data not shown). TGFß treatment for 2 days decreased the G1 phase and increased the S and sub-G1 phases. This sub-G1 phase (suggestive of apoptosis) was reduced in PRKAR1A inactivated cells (data not shown).
To investigate whether the TGFß pathway alteration due to PRKARIA inactivation affected TGFß-induced apoptosis, viable and apop- totic cells were studied by Annexin V/propidium iodide costaining.
Without treatment, siR1A did not change the proportion of cell phases (Fig. 5A/B, Control) but decreased TGFß-induced
decreases in viable cells via apoptosis (Fig. 5A/B). TGFB (5 ng/ml) for 48 hours decreased the percentage of viable cells to 71.44 ± 1.28% for cells transfected with siRIA and 65.84 ± 0.94% for siCtr cells (P < 0.05). The percentage of cells in late apoptosis was 9.55 ± 0.41% for siRIA versus 13.09 + 0.62% for siCtr (P < 0.05). siRIA was more protective at 50 ng/ml TGFB [viable cells: 58.67 ± 1.26% (siRIA) versus 39.25 ± 3.54% (siCtr); P < 0.05]. Cotransfection with siR1A and a SMAD3 expression vector (SMAD3-Flag) increased the level of apoptotic cells after TGFB treatment [siRIA: 33.55 ± 1% (SMAD3-Flag) versus 30.55 ± 0.7% (pCDNA3+); P < 0.05; Fig. 5C], indicating that the protective effect of PRKAR1A silencing on TGFß-induced apoptosis is due in part to decreasing SMAD3.
Finally, we tested the levels of caspase-3, a proapoptotic protein, and its targets, poly(ADP-ribose) polymerase (PARP) and lamin A/C. TGFB enhanced the accumulation of cleaved/active caspase-3, PARP, and lamin A/C in H295R cells (Fig. 5D), but siR1A reduces this cleavage.
Discussion
Inactivation of PRKAR1A by siRNA is an appropriate approach to study the consequences of PRKAR1A mutations observed in CNC (7), primary pigmented nodular adrenocortical dysplasia (PPNAD; ref. 15), and endocrine tumors (17). Indeed, the majority of these mu- tations lead to degradation of the mutant mRNA by NMD. Treatment of HEK293 or H295R cells with siR1A inactivates PRKAR1A and alters PKA activity in a manner that is not counteracted by compensatory regulation of other PKA subunits (32, 33). This is the first report using siRNA to inactive PRKAR1A in adrenal cells, increasing PKA activity as occurs in tumors from CNC patients (7), in prkar1a knockout mouse embryos (34), and in fibroblast cells with a prkar1a homo- zygous deletion (35). PRKAR1A inactivation increases both basal and forskolin-induced CRE transcriptional activity as well as the endog- enous target gene expression NURR1. Thus, the cellular consequen- ces of PRKAR1A inactivation might be modulated by the tone of the cAMP pathway. This stimulation of cAMP-dependent transcription could be blocked by H89, indicating that it is mediated by PKA.
A
Control
TGFB 5 ng/ml
TGFß 50 ng/ml
C
ns
r
LA
LA
LA
40
Propidium iodide staining
siCtr
Apoptotic cells (%)
V
EA
V
EA
V
EA
35
*
Late apoptotic
-
Early
LA
LA
LA
T
Viable apoptotic
30
SİR1A
V
EA
V.
EA
V
EA
Protein
25
4nonspecific
+S3-Flag
Annexin V staining
SMAD3 Flag
SMAD3 Flag
+B-actin
B
pCDNA3+
pCDNA3+
*
siCtr
SİR1A
80
ns
*
TGFß
Cells (%)
60
*
40
D
I
*
*
H295R
20
ns
ns
Ctrl
TGF₿
ns
*
siCtr siR1A siCtr siR1A
0
V
EA
LA
V
EA
LA
V
EA
LA
Cleaved
Caspase-3
Control
TGFB 5 ng/ml
TGF 50 ng/mL
Cells (%)
Cleaved PARP
*
*
V
76.67±0.89
V
65.84±0.94
V
39.25±3.54
76.56+0.28
71.44±1.28
58.67±1.26
EA
16.51±0.89
Cleaved Lamin A/C
EA
21.07±0.51
*
28.34±1.03
17.05±0.18
19.01±0.94
EA
21.15±1.05
LA
6.82±0.03
*
LA
13.09±0.62
*
LA
32.41±2.71
ß-actin
6.39±0.30
9.55±0.41
20.18±1.24
cAMP/PKA pathway
Ligand 1000
Inactivation of the Carney complex gene 1 (PRKAR1A)
TGFß pathway TGFß
Gsa
AC
R R
CAMP
CC
R1A
R1A
SMAD3
P
SMAD3
2
R
R
1
P
SMAD4
SMAD3 SMAD4
C
C
C
C
/ PKA activity
4
3
Tumorigenesis
Apoptosis
The cellular mechanisms linking cAMP pathway dysregulation to endocrine tumorigenesis are still poorly understood despite numerous observations of activating mutations of receptors or G protein and inactivating mutations of phosphodiesterases or PRKARIA in human tumors (36-38). The role of the TGFß pathway in adrenal cortex physiology led to the hypothesis that PRKAR1A inactivation might alter TGFß signaling, as there is cross-talk between cAMP and TGFß pathways. In keratinocytes and in cortical cells, the cAMP pathway abolishes the interaction of SMAD3 with the transcriptional coactivators CREB-binding protein and p300 (24, 25). Here, PRKAR1A inactivation inhibits the mRNA and protein expression of SMAD3, a major distal component of the TGFß pathway. This SMAD3 decrease reduced the TGFß reporter response in HEK293 cells and decreased SERPINE1 expression in response to TGFß via PKA. This study shows in vitro as well as in vivo the inhibition of SMAD3 by cAMP and ACTH in the adrenal cortex. This differs from the gonads and reprogrammed tumor cells from pluripotent adrenocortical stem/progenitors adopting a gonad-like differentiation after inhibin & inactivation (39, 40). This difference also underlines the tissue-specific effect of cAMP pathway dysregulation in tumorigenesis. How the stability of SMAD3 protein is regulated is not fully elucidated. The role of the ubiquitin-proteasome system has been suggested (39, 40). The regulatory subunits of PKA and SMAD3 physically interact via A-kinase anchoring proteins (41, 42).
TGFß regulates adrenal development (22, 23) and can inhibit adrenal steroid production (43, 44). TGFß can act either as a tumor suppressor or promote cancer progression. In human adrenocor- tical cells, TGFß treatment decreases cell proliferation (45). Here, TGFß altered the cell cycle and induced apoptosis in adrenocor- tical cells (this article and ref. 30). TGFß-regulated apoptosis
is cell type and context dependent. TGFß-regulated genes could mediate these apoptotic effects (46, 47). Here, PRKAR1A silencing protected H295R cells from TGFß-induced apoptosis and reduced the cleavage of caspase-3, PARP, and lamin A/C.
In conclusion, the cAMP/PKA pathway is an important signaling pathway for adrenocortical physiology. Understanding how dysre- gulation of this pathway in adrenocortical tumors can modulate other signaling pathways is key for understanding tumorigenesis. Our results show for the first time that cAMP/PKA pathway dysregulation after PRKARIA inactivation inhibits the TGFB pathway in adrenocortical tumor cells. This new mechanism of TGFß inhibition, mediated by PKA-dependent SMAD3 inhibition, protects adrenocortical cells from the apoptotic effect of TGFB (Fig. 6). This finding is consistent with TGFß pathway acting as a tumor suppressor in human cancer (48-50) and points to potential new targets in the treatment of these neoplasms.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Acknowledgments
Received 5/4/09; revised 7/6/09; accepted 7/13/09; published OnlineFirst 9/8/09.
Grant support: Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, Carney Complex Network (ANR-08-GENOPAT- 007), Programme Hospitalier de Recherche Clinique (PHRC060251), and Réseau COMETE (PHRC AOM 06). B. Ragazzon was recipient of fellowships from Fondation de la Recherche Médicale and the Conny-Maeva Foundation.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Dr. Franck Verrecchia for providing us SMAD3-Flag vector and the members of the FACS Core Facility of Institute Cochin.
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7284
Cancer Research The Journal of Cancer Research (1916-1930) | The American Journal of Cancer (1931-1940)
AAGR American Association for Cancer Research
Inactivation of the Carney Complex Gene 1 (Protein Kinase A Regulatory Subunit 1A ) Inhibits SMAD3 Expression and TGF B-Stimulated Apoptosis in Adrenocortical Cells
Bruno Ragazzon, Laure Cazabat, Marthe Rizk-Rabin, et al. Cancer Res 2009;69:7278-7284. Published OnlineFirst September 8, 2009.
Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-09-1601
Supplementary Material
Access the most recent supplemental material at: http://cancerres.aacrjournals.org/content/suppl/2009/09/04/0008-5472.CAN-09-1601.DC1.ht ml
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