Teratocarcinoma-Derived Growth Factor-1 Is Upregulated in Aldosterone-Producing Adenomas and Increases Aldosterone Secretion and Inhibits Apoptosis In Vitro
Aldosterone-producing adenomas (APA) are a frequent cause of secondary hypertension characterized by autonomous hypersecretion of aldosterone. However, the molecular mechanisms involved in adrenal tumorigenesis and deregulated aldosterone secretion are currently unknown. To identify putative functional genes, a transcriptional screening was performed on 8 APA and 3 normal adrenals (NA) using oligonucleotide microarrays. Data were next validated on an expanded set of samples by real-time PCR (APA, n=19; NA, n=10). The epidermal growth factor–like teratocarcinoma-derived growth factor-1 (TDGF-1) was upregulated in APA compared with NA (14.7-fold and 21.4-fold by microarray and real-time PCR, respectively). In vitro studies and Western blot analysis using the NCI H295R adrenocortical cell line showed that TDGF-1 increased Akt phosphorylation on Thr308 and Ser473, consistent with activation of phosphatidylinositol 3-kinase/Akt signaling, and also demonstrated a concomitant inactivation of the Akt substrate glycogen synthesis kinase-3β via Ser9 phosphorylation. Furthermore, TDGF-1 mediated a 3.8±0.4-fold increase in aldosterone secretion (n=4) that was specifically blocked by the phosphatidylinositol 3-kinase inhibitors wortmannin (50 nmol/L) and LY294002 (20 μmol/L). Finally, TDGF-1 protected H295R cells from apoptosis induced by staurosporine, causing a decrease in caspase-3 activity, a reduction in the inactivation of poly(ADP-ribose) polymerase, and an inhibition of DNA fragmentation, detected by the TUNEL reaction and fluorescence microscopy that was blocked by LY294002. Taken together, our data suggest that TDGF-1, which is significantly upregulated in APA and mediates aldosterone hypersecretion and deregulated growth in adrenocortical cells in vitro, may represent a key player in the development and pathophysiology of primary aldosteronism.
- aldosterone-producing adenoma
- NCI H295R cells
- teratocarcinoma-derived growth factor
- phosphatidylinositol 3-kinase/Akt signaling
Primary aldosteronism (PA) is the most frequent form of endocrine hypertension, accounting for up to 5% to 10% of all hypertensive patients,1 and is characterized by the chronic, excessive, and autonomous secretion of aldosterone by the adrenal gland. The diagnosis of this form of hypertension is fundamental because, compared with essential hypertensives with similar risk profiles, patients with PA are more prone to stroke and myocardial infarction2 and display an increase in cardiovascular damage and metabolic complications.3
Aldosterone-producing adenomas (APA) are a common underlying cause of PA and are found in 30% to 40% of PA patients, whereas bilateral adrenal hyperplasia is present in 60% to 70% of patients.4 Unilateral adrenalectomy normalizes, or at least markedly improves, the blood pressure in patients with APA, and therefore, APA is the most common, specifically treatable, and potentially curable form of hypertension.4,5 However, the molecular mechanisms underlying the alterations in cell growth in the adrenal cortex and the hypersecretion of aldosterone have not yet been defined.
Previous genomic analyses6–9 have provided evidence for differential gene expression in APA compared with normal adrenals (NA). The present study was undertaken to better define upregulated gene transcripts in highly selected APA and to investigate the functional role of such genes specifically with regard to autonomous aldosterone secretion and deregulated cell growth. The APA used in this study were removed from patients studied in our hypertension unit who had been homogeneously selected following a rigorous diagnostic flowchart that includes adrenal venous sampling and postadrenalectomy evaluation. Furthermore, strict criteria were used for adrenal venous sampling, cannulation, and lateralization,10 and finally, NA and APA were removed under identical conditions; that is, in both cases, the same technique, surgical team, and anesthetic was used.
We demonstrate here, by microarray analysis and real-time PCR, that teratocarcinoma-derived growth factor-1 (TDGF-1, Cripto-1, CR-1), which belongs to the epidermal growth factor–Cripto-FRL1-Cryptic (CFC) protein family and plays a key role in early vertebrate development and carcinogenesis,11–13 is a highly upregulated gene in APA.11 Furthermore, immunohistochemical studies show that this growth factor displays a distinct localization to APA nuclei, in contrast to the cytoplasmic expression observed in the surrounding adrenal cortex and in the zona glomerulosa of NA tissue. Functional studies of TDGF-1 in NCI H295R cells14 demonstrate that TDGF-1 mediates an activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway and both an increase in aldosterone secretion and an inhibition of apoptosis in vitro. Furthermore, these latter 2 effects are specifically blocked by PI3K inhibitors. Therefore, this study identifies a gene transcript upregulated in APA that displays functional roles in vitro that are relevant to the aldosterone hypersecretion and deregulated cell growth typical of an APA.
An expanded Methods section is available online at http://hyper.ahajournals.org.
RNA samples used for microarray experiments (n=12) were assayed for purity and quality using an Agilent Bioanalyzer. The gene expression profiling platform was oligonucleotide array Affymetrix GeneChip HG-U133 Plus 2.0. cRNA synthesis, labeling, hybridization, and chip scanning were performed following the manufacturer’s protocols. CEL files were analyzed using dChip 2008 software.15
RNA Extraction From Adrenal Tissues
Adrenal tissue (≈100 mg) was homogenized (Ultra-Turrax T8 IKA tissue homogenizer, Staufen, Germany) in 1 mL of TRI-reagent (Sigma-Aldrich Corp, St. Louis, Mo) and processed according to the manufacturer’s instructions.
Gene Expression Assays
Reverse transcription was performed using 5 μg of total RNA, 250 ng of random primers, and SuperScript II reverse transcriptase (Invitrogen Corp, Carlsbad, Calif), as described by the manufacturer. Real-time PCR was performed in triplicate using TaqMan gene expression assays (Applied Biosystems, Foster City, Calif) on an Applied Biosystems ABI 7500 instrument following standard protocols. Gene expression levels were analyzed using the 2−ΔΔCt relative quantification method, using β-actin as the endogenous reference gene.
Total RNA was extracted from 100-mm-diameter plates of subconfluent cells (RNeasy mini kit, Qiagen, Valencia, Calif). First-strand cDNA were generated from 5 μg of total RNA using Superscript II reverse transcriptase (Invitrogen) and used as templates for PCR amplification of TDGF-1 or GAPDH cDNA fragments.
PCR Cloning of TDGF-1
The full-length coding region of the TDGF-1 cDNA was PCR amplified from first-strand cDNA using pfu polymerase (Promega, Madison, Wis) from an APA expressing the highest level of the TDGF-1 transcript and inserted into pcDNA3.1. A control plasmid was constructed (pcDNA3.1/control) comprising TDGF-1 cDNA inserted in the antisense direction with respect to the cytomegalovirus promoter.
Frozen sections of APA or adjacent normal cortical adrenals (4 μm) were incubated for 1 hour with antihuman TDGF-1 polyclonal antibody (ab19917, Abcam, Cambridge, United Kingdom; 200 μL, 1:20 dilution). Bound primary antibody was visualized using the Envision system horseradish peroxidase–labeled polymer (Dako, Carpinteria, Calif) conjugated with anti-rabbit secondary antibody and staining completed with 3,3′-diaminobenzidine substrate-chromogen. Nuclei were counterstained with hemalum.
NCI H295R human adrenocortical carcinoma cells14 were cultured in RPMI 1640 medium without phenol red (Gibco, Billings, Mont) supplemented with 2% FBS, insulin-transferrin-selenium A, l-glutamine (292 μg/mL), streptomycin (100 μg/mL), and penicillin (100 U/mL) (all from Gibco) and maintained at 37°C in a humidified 5% CO2 incubator.
Transfection of H295R Cells
H295R cells (2×106) were electroporated using 2 μg of plasmid in Nucleofector solution R (100 μL) with the Amaxa nucleofector (program P20, Amaxa Biosystems, Cologne, Germany).
Cell Proliferation Assay
Cell proliferation of transfected H295R cells was determined using the Cell Titer 96 AQueous One solution cell proliferation assay kit (Promega).
Poly(ADP-Ribose) Polymerase Assay
Transfected H295R cells were incubated with or without staurosporine (STS; 500 nmol/L for 16 hours) and then washed, lysed, and sonicated in SDS-PAGE sample buffer (300 μL). Samples (15 μL) were resolved by 10% SDS-PAGE, and poly(ADP-ribose) polymerase (PARP), was visualized by Western blotting.17
Cells were transfected and apoptosis was induced as described for the PARP assay. Caspase-3 activity was quantified in cell extracts (50 μg), using a colorimetric assay kit (CaspACE assay system, Promega) as described previously.17
Transfected cells were grown in Laboratory-Tek Chamber Slides (Sigma-Aldrich), and apoptosis was induced with STS (500 nmol/L) with or without LY294002 (20 μmol/L). The TUNEL reaction was performed according to the manufacturer’s protocol (DeadEnd fluorometric TUNEL system, Promega). Slides were mounted in Vectashield with 4′,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, Calif) and viewed by fluorescence microscopy (Zeiss, Axio Observer, Göttingen, Germany).
A chemiluminescent substrate was used to visualize specific bands binding horseradish peroxidase–linked secondary antibodies (Pierce Biotechnology).
SAS software, version 8 (SAS Institute Inc., Cary, NC), was used for statistical analyses. Data are expressed as mean±SD, and differences between variables were evaluated using a parametric test for repeated measures and a Bonferroni post hoc test. A probability of less than 0.05 was considered statistically significant.
Microarray Analysis of APA Transcript Profiles and Upregulation of TDGF-1 in APA
Oligonucleotide microarrays were used to perform a transcriptional screening of 8 APA, compared with 3 NA. On the basis of the filtering algorithm, described in the Methods, 64 probe sets (corresponding to 53 genes) were found to be differentially expressed in APA compared with NA (33 upregulated, 20 downregulated), and TDGF1 ranked as the top upregulated gene (average 14.7-fold upregulation). Supplemental Tables I and II list the upregulated and downregulated known genes, respectively. The hierarchical clustering of differentially expressed genes is presented in Figure 1. Interestingly, the transcriptional fingerprint of a single unilateral adrenal hyperplasia sample corresponded closely to that of NA but not to that of APA.
The expression level of TDGF1 was further examined by real-time PCR on a broader set of adrenal samples (19 APA and 10 NA) comprising 5 APA and 2 NA already used for the microarray screening. TDGF1 was also upregulated in this group of APA (Figure 2A) and was observed in 79% of the APA samples (n=15/19, 21.4-fold compared with NA, Table 2). The microarray data were also confirmed for 3 other selected genes (TPD52, VSNL1, and IGSF4), thus validating our gene expression platform (Figure 2B through 2D, Table 2). Finally, the expression of TDGF1 was assayed on distinct pathological adrenal samples, including cortisol-producing adenomas from patients with Cushing syndrome (n=3), adrenal incidentalomas (n=3), an aldosterone-producing carcinoma, and a primary adrenal hyperplasia. In all these samples, TDGF1 expression levels were comparable to those present in NA (data not shown). Thus, the upregulation of TDGF1 appeared as a primary feature of APA and not of other adrenal diseases.
TDGF-1 Is Localized to the Zona Glomerulosa in NA Glands
Immunohistochemical staining of frozen tissue sections demonstrated that TDGF-1 was expressed in the cytoplasm of NA restricted to the zona glomerulosa (Figure 3A); in contrast to the immunoreactivity observed in APA, which was localized to nuclei but cytoplasmic in the surrounding adrenal cortex (Figure 3B). Nuclear and cytoplasmic immunoreactivity was confirmed in the pancreas control specimen that was undetectable in the negative control (data not shown).
Human Adrenocortical Cells (NCI H295R) as a Model Cell Line to Study the Function of TDGF-1
TDGF-1 was undetectable in H295R cells by semiquantitative RT-PCR and in a pooled sample of first-strand cDNA from NA, whereas a TDGF-1-specific fragment was amplified from a pooled sample of first-strand cDNA from APA (Figure 4); the absence or low level expression of TDGF-1 gene transcription indicates the suitability of this cell line to study TDGF-1 function in vitro.
TDGF-1 Activates the Akt Signaling Pathway in H295R Cells
TDGF-1 has been reported previously to activate PI3K/Akt signaling in diverse cell types.11,18,19 H295R cells were transfected with either pcDNA3.1/TDGF-1 or pcDNA3.1/control, and Western blot analysis demonstrated a greater activation of Akt, revealed by increased phosphorylation of Akt residues Ser473 and Thr308, in cell lysates from TDGF-1-transfected H295R cells compared with controls (Figure 5). Furthermore, increased phosphorylation and therefore inactivation of the Akt substrate glycogen synthase kinase-3β on residue Ser9 was observed in TDGF-1-transfected H295R cells (Figure 5).
TDGF-1 Mediates an Increase in Aldosterone Secretion From H295R Cells That Is Specifically Blocked by PI3K Inhibitors
H295R cells were transfected with pcDNA3.1/TDGF-1 and basal aldosterone secretion was measured by radioimmunoassay and compared with that of cells transfected with pcDNA3.1/control. Aldosterone secretion was significantly increased (P<0.005) from TDGF-1-transfected cells, displaying a 3.8±0.4-fold increase in secreted aldosterone after 72 hours (Figure 6A).
The increased aldosterone secretion was not due to an increase in cell proliferation (1.06±0.07-fold change in cell proliferation after 72 hours for cells transfected with TDGF-1/control plasmid, n=4, ±SD).
We investigated the effect of angiotensin II (Ang II) and TDGF-1 on aldosterone secretion. Ang II (10 nM) produced a 13.7±1.2-fold increase in aldosterone secretion in control-transfected cells compared with unstimulated cells and a 15.8±1.6-fold increase in TDGF-1-transfected cells. TDGF-1 mediates a 3.8-fold increase in aldosterone secretion in unstimulated cells, and therefore, TDGF-1 does not potentiate the effect of Ang II in H295R.
The possible involvement of PI3K in TDGF-1-mediated aldosterone secretion was studied using specific inhibitors (50 nmol/L wortmannin and 20 μmol/L LY294002), which decreased the TDGF-1-mediated increase in aldosterone secretion (Figure 6B) to levels similar to that of untreated control cells (1.1±0.1-fold and 1.3±0.2-fold increases in aldosterone secretion in the presence of LY294002 and wortmannin, respectively; both values not significantly different from untreated control).
H295R cells transfected with TDGF-1 exhibited no significant difference in cell proliferation compared with control cells after 72 hours (0.99±0.04-fold change, n=4), and no significant differences in cell proliferation were detected in cells transfected with either TDGF-1 or the control plasmid after 72 hours in the presence of either wortmannin (50 nmol/L) or LY294002 (20 μmol/L), 1.02±0.06 and 0.97±0.02-fold change in cell proliferation in control cells in the presence of LY294002 and wortmannin, respectively, and 1.01±0.04 and 0.97±0.02-fold change in TDGF-1-transfected cells in the presence of LY294002 and wortmannin, respectively (n=4).
TDGF-1 Protects H295R Cells From Apoptosis, and This Effect Is Blocked by a PI3K Inhibitor
TDGF-1 has been reported to function as a survival factor with an antiapoptotic effect in human cervical carcinoma cells via a PI3K-dependent signaling pathway.19 Induction of apoptosis by STS and specific detection of PARP by Western blotting demonstrated that cleavage of catalytically active PARP was relatively protected in TDGF-1-transfected cells: Figure 7A shows an increased ratio of the uncleaved:cleaved forms of PARP in these cells; thus, the inactive, cleaved form of PARP, a hallmark of apoptosis, is relatively more abundant in control cells.
In addition, the increase in caspase-3 activity in response to STS was markedly reduced in cells transfected with TDGF-1: a 19.7±3.7-fold increase in caspase-3 activity in control cells (caspase-3 activity in STS-stimulated/unstimulated cells) compared with a 3.6±0.6-fold increase in caspase-3 activity in TDGF-1-transfected cells (Figure 7B).
The possible involvement of PI3K in TDGF-1-mediated decreased sensitivity to apoptotic stimuli in H295R cells was studied using the TUNEL assay. In agreement with an antiapoptotic effect of TDGF-1, the strong fluorescent labeling observed in the nuclei of control cells was greatly decreased in TDGF-1-transfected cells. Furthermore, in the presence of the PI3K inhibitor LY294002 (20 μmol/L), this protective effect of TDGF-1 toward apoptotic stimuli was partially blocked (Figure 8).
PA is the most common form of endocrine hypertension, and APA comprises a frequent subtype of this disease (30% to 40% of the total). The removal of the adenoma in patients with APA normalizes the excessive aldosterone secretion, characteristic of this condition, and, as a consequence, cures or markedly improves the hypertensive status.
This work defines TDGF-1 as a highly upregulated gene in APA compared with NA tissue. In agreement with previous transcriptional screens, we observed an increased expression of both aldosterone synthase (CYP11B2) and the 5-hydroxytryptamine receptor 4 (HTR4)6,7,9; however, we did not find evidence for elevated expression of either the luteinizing hormone receptor8 or the Ca2+/calmodulin-dependent protein kinases.9 This could be due to heterogeneity of the APA phenotype or to differences in normal subjects and patient selection: for example, in this study, APA were used from patients with florid forms of PA, adrenalectomized after performing adrenal venous sampling using particularly strict criteria both for successful cannulation and lateralization.
This study assigns a functional role for TDGF-1 in aldosterone secretion and in the protection from apoptosis in human adrenocortical carcinoma cells (NCI H295R). We show that inactivating PI3K with specific inhibitors blocks the increase in aldosterone secretion mediated by TDGF-1 in H295R cells and at least partially blocks the reduction in sensitivity to apoptosis. PI3K activation is followed by Akt phosphorylation, a key effecter of PI3K-mediated cell survival.20,21 The present study demonstrates that the cell survival effect and the increase in aldosterone secretion are associated with the activation of Akt by increased phosphorylation in the C-terminal regulatory domain on residue Ser473 and in the catalytic domain on residue Thr308. Consistently, an increase in Akt signaling was demonstrated further by the enhanced Ser9 phosphorylation of the Akt substrate glycogen synthase kinase-3β.
The antiapoptotic effect of TDGF-1 associated with Akt activation is consistent with the known effect of Akt in promoting cell survival via the phosphorylation and inactivation of the proapoptotic protein Bad.22 In agreement with the present study, other groups have demonstrated that TDGF-1 activates PI3K/Akt signaling, for example, in mouse mammary epithelial cells18 and in cervical carcinoma cells, leading to the phosphorylation and inactivation of glycogen synthase kinase-3β19; this latter study also demonstrated an antiapoptotic effect of TDGF-1 that was decreased by a specific PI3K inhibitor. Furthermore, the suppressed tumor growth in vivo, resulting from antibody blockade of TDGF-1,12,23 was related to increased apoptosis mediated by c-Jun-N-terminal kinase and inhibition of Akt phosphorylation.23
Other factors have been identified that increase aldosterone secretion from adrenocortical cell lines or from cells isolated from the zona glomerulosa: for example, sphingosine-1-phosphate stimulates aldosterone secretion via the PI3K/Akt and mitogen-activated protein/extracellular signal–regulated kinase pathways24; an adipokine, CTRP1, expressed specifically in the zona glomerulosa of the adrenal cortex, increased aldosterone production in H295R cells.25 Furthermore, the phosphoprotein Disabled-2, expressed in the zona glomerulosa and zona intermedia of the rat adrenal gland, increased aldosterone secretion, although under conditions of Ang II stimulation.26 However, to our knowledge, this is the first study that in addition to identifying a gene transcript specifically overexpressed in APA, also demonstrates potentially relevant functional roles in the adrenal cell model, NCI H295R, and provides evidence for the underlying mechanism via PI3K/Akt signaling.
TDGF-1 functions as an essential cofactor for signaling by the transforming growth factor-β family ligand Nodal during early embryogenesis and belongs to the epidermal growth factor–CFC family of proteins that are present in diverse species such as zebrafish, Xenopus, and mouse, as well as humans.27 TDGF-1 is overexpressed in many human cancers, such as breast, pancreatic, ovarian, and colon cancers. Furthermore, a functional role for TDGF-1 in tumor growth is suggested because its expression can transform some mammary cell lines; in addition, antibody blockade of TDGF-1 suppresses tumor growth in vivo,12,23 and antisense inhibition of TDGF-1 results in the loss of the transformed phenotype of colon carcinoma cells.28 In normal adult tissues, TDGF-1 is primarily expressed in mammary glands during pregnancy and lactation; this is the first report of TDGF-1 expression in the NA gland and in APA. TDGF-1 is primarily distributed as a plasma membrane–bound protein but can also exist in a soluble form, with a dual function as both receptor and growth factor: however, we show here that, in NA cells, TDGF-1 has a cytoplasmic location, in contrast to the pathological state in APA, where TDGF-1 is localized exclusively to the nucleus. Nonetheless, a cytoplasmic subcellular distribution for TDGF-1 has indeed been reported previously.29,30 The reason for the nuclear versus cytoplasmic localization of TDGF-1 in APA or normal tissue, respectively, is unclear. It could be proposed that TDGF-1, in addition to its well-characterized role as a coreceptor for Nodal in early embryogenesis, may undergo nuclear translocation under certain conditions in adult tissues and subsequently regulate gene expression, thereby mediating other cellular processes. Such a precedent has been described previously. For example, epidermal growth factor receptor family members, in addition to the traditional epidermal growth factor receptor pathway involving transduction of mitogenic signals via the activation of signaling cascades, can also translocate to the nucleus and regulate gene expression; a number of other receptor tyrosine kinases and cytokine receptors also undergo similar nuclear localization,31 but further studies would be required to establish whether this is the case for TDGF-1.
Future studies should address whether the upregulation of TDGF-1 is specific to a particular subtype of APA (eg, Ang II responsive/unresponsive). The number of Ang II–responsive patients in the present study was too small to adequately answer this question.
This work defines TDGF-1 as a highly upregulated gene in APA compared with NA cortex and assigns a functional role for TDGF-1 in aldosterone secretion and in the protection from apoptosis in human adrenocortical carcinoma cells via PI3K/Akt signaling. Changes in the level of TDGF-1 expression in specific cell populations of the adrenal gland, that is, in the zona glomerulosa, may constitute one of several changes that are important in the pathogenesis of an APA, in which the cumulative effect of a number of genetic alterations is required for the initiation of APA formation and subsequent autonomous hypersecretion of aldosterone.
The present study provides evidence for a potential role of TDGF-1 in the pathogenesis of APA and highlights a dual function for this epidermal growth factor–like protein in the inhibition of apoptosis and in increasing aldosterone secretion from adrenal cells via deregulated Akt signaling. In addition, we demonstrate significant upregulation or downregulation of a number of other genes compared with NA, thus providing the basis for future studies that address the role of genomic alterations in the pathophysiology of APA.
Sources of Funding
This study was supported by grants from the Italian Ministry of University, Scientific and Technological Research (COFIN No. 2006060985), Fondi Regione Piemonte Ricerca Finalizzata 2007, MUR (ex 60%), and Compagnia di San Paolo.
- Received January 13, 2010.
- Revision received January 28, 2010.
- Accepted March 17, 2010.
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