- Email: [email protected]
Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor
Accepted Manuscript Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor Carolin Schwafertz, Sven Schinner, Markus C. Kühn, Matthias Haase, Amelie Asmus, Birgit Mülders-Opgenoorth, Ishrath Ansurudeen, Peter J. Hornsby, Henning Morawietz, Elke Oetjen, Matthias Schott, Holger S. Willenberg PII:
S0303-7207(16)30484-1
DOI:
10.1016/j.mce.2016.11.015
Reference:
MCE 9729
To appear in:
Molecular and Cellular Endocrinology
Received Date: 20 June 2016 Revised Date:
17 November 2016
Accepted Date: 20 November 2016
Please cite this article as: Schwafertz, C., Schinner, S., Kühn, M.C., Haase, M., Asmus, A., MüldersOpgenoorth, B., Ansurudeen, I., Hornsby, P.J., Morawietz, H., Oetjen, E., Schott, M., Willenberg, H.S., Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.11.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Schwafertz / Willenberg et al.,
β-catenin and bFGF – graphical abstract
ACCEPTED MANUSCRIPT
Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
1
1
1
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Short Title:
1
1
Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
2
5
1,2
,
2,3
, Peter J.
1
Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S. 1,6
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Willenberg
Graphical Abstract
Endothelial cell products are capable in regulating SF-1, StAR and CITED2, essential factor for adrenal organogenesis or function. However, the signalling involved was net yet conclusively described. Schwafertz et al. have found that β-catenin-mediated transcription is involved in the process and that endothelial cell products do not use the classical wnt-signalling pathway. The secretion of bFGF by endothelial cells partly explains the effects.
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Fig. 6
Schwafertz / Willenberg et al.,
β-catenin and bFGF – Title page
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Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
1
1
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Short Title:
1
1
1
Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
2
5
2,3
1,2
,
, Peter J.
1
Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S.
Affiliations:
1
1,6
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Willenberg
Division for Specific Endocrinology, Medical Faculty, Heinrich-Heine
University Dusseldorf; D-40225 Dusseldorf;
2
Department of Medicine III, Carl
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Gustav Carus Medical School, University of Technology, D-01307 Dresden, 3 Germany; Department of Molecular Medicine and Surgery, L1:01 Rolf Luft 4
Centrum, Karolinska Institute, Stockholm, Sweden; Department of Physiology and Sam and Ann Barshop Institute for Longevity and Aging 5
Studies, University of Texas Health Science Center, San Antonio, TX, USA; 6 Institute of Pharmacology, University of Göttingen; Germany; Division of Endocrinology and Metabolism, Rostock University Medical Center, Germany Holger S. Willenberg
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Correspondence to:
Division of Endocrinology and Metabolism Rostock University Medical Center Ernst-Heydemann-Str, 6, D-18057 Rostock, Germany
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Tel: +49 (381) 494-7521; Fax: +49 (381) 494-7522 eMail: [email protected]
The plasmid for StAR Promoter was a generous gift from Dr. D. Stocco
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Acknowledgement:
(Texas Tech University Health Sciences Center, Lubbock, USA). This work was supported by the Doktor Robert Pfleger-Stiftung, Bamberg, Germany (HSW).
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Abstract
Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of β-
catenin activity in adrenocortical NCI-H295R cells.
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catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect β-
Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of β-catenin and an increase in β-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway.
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Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced β-catenindependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM.
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Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate β-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.
aldosterone; adrenal gland; beta-catenin; basic fibroblast growth factor; CITED2
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Keywords:
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1. Introduction
The adrenal gland is an effector organ of the hypothalamo-pituitary-adrenal axis and the reninangiotensin system. As such, it is involved in the regulation of salt and water homeostasis, extracellular volume balance, blood pressure, immune function, and energy metabolism in response to stress.
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The adrenal gland is heavily vascularized and almost every adrenocortical cell is in direct contact to an endothelial cell, whereby the microstructure is of relevance for proper adrenal function (Dobbie et al. 1966, Hornsby et al. 1987, Ehrhart-Bornstein et al. 1998). The regulation of adrenal blood flow is dependent on corticotropin (ACTH) (Vinson et al. 1985, Breslow et al. 1992, Thomas et al. 2003, Mohn et al. 2005, Zhang et al. 2007) and endothelial cell products affect the synthesis of adrenocortical
Ehrhart-Bornstein et al. 1998, Willenberg et al. 2008).
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hormones, including aldosterone (Vinson et al. 1985, Hinson et al. 1991, Nussdorfer et al. 1997,
Although a number of specific endothelial cell-derived factors have been identified, the overall effect of
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endothelial cell-derived products on adrenocortical cells has been poorly studied. Endothelial cellconditioned medium (ECCM) is a cocktail of factors, containing endothelin-1, angiotensin-2 (Kifor et al. 1987, Vane et al. 1993), interleukin-6 (Willenberg et al. 2008) and other, unidentified proteins (EhrhartBornstein et al. 1998, Willenberg et al. 2008). The aforementioned known components of the ECCM do not explain the effects observed when adrenocortical cells were incubated with ECCM (Ansurudeen et al. 2006, Ansurudeen et al. 2007, Willenberg et al. 2008, Haase et al. 2009, Ansurudeen et al. 2009, Paramonova et al. 2010). However, Rosolowski et al., described an endothelium-derived 3000
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Da protein which leads to increased aldosterone concentrations in supernatants of cultured bovine adrenal cells via the protein kinase C pathway (Rosolowsky et al. 1994). Interestingly, our studies have shown that the endothelial cells act on adrenocortical cells involving proteins essential for adrenocortical development, including steroidogenic factor (SF)-1, steroidogenic
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acute regulatory protein (StAR) and CITED2 (Ansurudeen et al. 2007, Haase et al. 2009). In addition, endothelial cell-derived products influence the proliferation of adrenocortical cells (Paramonova et al. 2010).
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Recently, proliferation and tumorigenesis of adrenocortical adenomas were demonstrated to be associated with activation of β-catenin (Tissier et al. 2005, Stratakis et al. 2007, Schinner et al. 2009, Berthon et al. 2010, Gaujoux et al. 2011). Furthermore, knock-down of β-catenin leads to a decrease in SF-1 positive cells as well as to an early disappearance of adrenocortical and adrenomedullary precursor cells (Kim et al. 2008). It was shown by other groups and ours that adrenocortical cells express frizzled receptors and βcatenin in the human adrenal gland and that activation of the Wnt signalling pathway stimulates aldosterone and cortisol biosynthesis through β-catenin (Chen et al. 2006, Schinner et al. 2007, Tadjine et al. 2008, Schinner et al. 2009). We therefore hypothesized whether ECCM could regulate βcatenin-dependent transcription and if so whether the Wnt signalling pathway is involved in its regulation. We here show that ECCM significantly up-regulates β-catenin-dependent transcription through MAP- and RTK-pathways.
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2. Materials and methods
This study aimed at better characterization of the ECCM effect on adrenocortical cells that was seen in previous studies whereby data on the adrenal hormonal responses and adrenal cell viability have been made available to the public (Ansurudeen et al. 2006, Ansurudeen et al. 2007, Ansurudeen et al.
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2009, Haase et al. 2009, Paramonova et al. 2010). Further experiments were conducted as follows.
2.1 Cell cultures
For our in vitro studies, three cell lines were used. NCI-H295R cells were cultured in DMEM/F12Glutamax medium (Invitrogen, Karlsruhe, Germany), containing fetal bovine serum (2 %, Invitrogen),
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insulin (66 nM, Sigma-Aldrich, Munich, Germany), hydrocortisone (10 nM, Sigma-Aldrich), apotransferrin (10 µg/mL, Sigma-Aldrich), β-estradiol (10 nM, Sigma-Aldrich), sodium selenite (30 nM, Sigma-Aldrich), penicillin (100 U/mL, Invitrogen) and streptomycin (100 mg/mL, Invitrogen), at 37°C in
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a humidified atmosphere of 95 % air, and 5 % CO2, as described previously (Ansurudeen et al. 2007). Primary adrenocortical cells were established from an adrenal gland of a patient who had undergone unilateral nephrectomy for renal cancer. An immortalized as described previously (Suwa et al. 2001), using pLEGFP-CMV-SV40T
and pBabe-hTERT
retroviral vectors to produce immortalized
adrenocortical cells. Immortalized adrenocortical (IMAC) cells were cultured in DMEM/F12-Glutamax (Invitrogen) with 10 % fetal bovine serum (Invitrogen), penicillin (100 U/ml, Invitrogen) and streptomycin (100 mg/ml, Invitrogen) at 37°C in a humidified atmosphere of 95 % air, and 5 % CO2 for
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further experiments (Werminghaus et al. 2014).
At confluency, H295R and IMAC cells were subcultured using Accutase (PAA Laboratories, Cölbe, 6
2
Germany) and seeded at density of 2×10 cells in 75-cm flasks. For transfection and protein extraction, cells were plated in 24-well culture plates. At confluency of 80%, cells were used for
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experimental procedures.
Different preparations of human umbilical vein endothelial cells (HUVEC) were used and obtained from normal term and normal pregnancy donors. HUVECs have been isolated using the following
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protocol (Ansurudeen et al., 2007). After being emptied of blood by perfusion with phosphate buffer (50 mM, pH 7.0), 15-cm-long segments of umbilical cord venous or arterial vessels were filled with collagenase I (1 mg/mL of endothelial cell medium, PromoCell, Heidelberg, Germany) and incubated at 37°C for 30 min. Cells were collected from the suspension by centrifugation at 50 g for 5 min at 4°C. The cell pellet was suspended and cultured in endothelial cell basal medium (PromoCell), containing the ingredients of the supplement pack (PromoCell), fetal calf serum (10 %) and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL), at 37°C in a humidified atmosphere of 95 % air and 5% CO2. For experiments, cells from passages 3 – 5 were used. Commercially available HUVEC and HUAEC preparations (Lonza, Walkersville, USA) were also used. At a confluency of over 80 %, fresh DMEM/F12 glutamax medium with the supplemental pack was added to endothelial cells and incubated for 48-72 hours to obtain ECCM. After this incubation period, ECCM was centrifuged to remove potential contaminat cells and stored at –20°C until further use.
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2.2 Plasmids and transfections The plasmid TOPFLASH reporter gene (with multiple binding sites for TCF/LEF-transcription factors which are co-activated by β-catenin) and the FOPFLASH reporter gene (with mutated TCF/LEFbinding-sites) are commercially available at UPSTATE biotechnology (Lake Placid, USA). The plasmids pT81 for studies of cAMP-dependent protein kinase A transcription as well as the 1300 StAR-Luc plasmid were used as described previously (Oetjen et al. 1994, Caron et al. 1997). Cells 5
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were seeded at a density of 2×10 cells per well in 24-well culture plates. After 24 h, when confluency exceeded 50 %, transfection was performed with Nanofectamin (PAA Laboratories, Cölbe, Germany) according to the manufacturer's protocol. The plasmids TOPFLASH, FOPFLASH, StAR-Luc and pT81 plasmids were transfected at a concentration of 0.5 µg per well along with the renilla luciferase pRLTK-Luc plasmid (Promega, Mannheim, Germany) at a concentration of 0.1 µg for internal control.
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24 h later, the cells were stimulated with DMEM/F12 basal medium or ECCM, alongwith various inhibitors or proteins for next 24 h. The NCI-H295R cells and the immortalized cells were treated with different concentrations of ECCM diluted in DMEM/F12 (5 %, 10 %, 20 %, 25 %, 30 %, 50 %, 75 %,
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and 100 %). The effect of Wnt signaling in NCI-H295R cells was studied in the presence of inhibitors of the Wnt signaling pathway using sFRP-1 (secreted frizzled-related protein-1; 10 ng/mL, R&D Systems, Inc., Minneapolis, USA) which interacts with Wnt ligands and impedes their binding to one of the frizzled receptors, or Dkk-1 (Dickkopf-related protein 1; 1 µg/mL, R&D Systems, Inc.) which binds to the LRP5/6 co-receptor and inhibits signal transduction. The role of protein kinases in NCI-H295R cells were studied using inhibitors of protein kinase A (H89, 10 µM, Calbiochem Merck, Darmstadt, Germany), protein kinase B (Akt inhibitor VIII, 1 µM, Calbiochem Merck), protein kinase C
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(bisindolylmaleimide I, 3 µM, Calbiochem Merck), MEK inhibitor (U0126, 25 µM, Promega), PI3-kinase inhibitor (Ly294002, 50 µM, Sigma-Aldrich) and genistein (inhibitor of tyrosine-specific protein kinases, 50 µM, Sigma-Aldrich). Briefly, NCI-H295R cells were incubated with 50 % ECCM with or without inhibitors and studied. Other stimulation experiments in NCI-H295R cells were performed with 50 %
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ECCM, including the bFGF receptor inhibitor Su5402 (22 µM, Merck) or bFGF (PromoCell, Heidelberg, Germany) at concentrations of 1 ng/mL or 10 ng/mL. Transfection experiments were also carried out with the StAR reporter gene in H295R cells, treated with 10 %, 25 % or 50 % ECCM as
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described previously (Paramonova et al. 2010). After the stimulation period, analyses were performed with the dual-luciferase reporter assay system (Promega) as described previously (Haase et al. 2009). Results are expressed as means ± standard deviation (SD) of a minimum of three independent experiments. Statistical analysis was performed with a t-test using Prism 4.1 (Graph Pad, San Diego, CA, USA).
2.3 Nuclear extraction and Western blot NCI-H295R cells were seeded in 6-well culture plates in DMEM/F12 medium. Cells were treated with 50% ECCM and U0126 (25 µM) or Ly294002 (50 µM) for 24h. For total protein extraction cells were lysed with lysis buffer 1 (10 mM HEPES pH 7.9; 10 mM KCl; 0.1 mM EDTA) containing 1 % Nonidet P40. For nuclear extraction, cells were treated first with lysis buffer 1 and then with lysis buffer 2 (20 mM HEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA). Concentrations of the extracted proteins were determined using the BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA).
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Denatured proteins were resolved by SDS-polyacrylamide gel electrophoresis, followed by transferring the proteins onto a nitrocellulose membrane. The blots were incubated with specific rabbit anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution, bovine secondary antibody (Dako Cytomation, Glostrup, Denmark) at a 1:5000 dilution and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) at a 1:1000 dilution for normalization. Protein detection was performed with chemiluminescence using ECL
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Plus Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) after incubation with HRP-Streptavidin (Jackson ImmunoResearch Laboratories, Suffolk, UK).
2.4 Reverse transcriptase-PCR (RT-PCR)
RNA was extracted from NCI-H295R cells and HUVEC using RNeasy MiniKit (Qiagen, Hilden,
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Germany) following the manufactor's instructions (Haase et al. 2009). Synthesis of cDNA was performed using the Ready-to-go t-primed first-strand kit (GE Healthcare, Buckinghamshire, UK). For amplification and detection of PCR products we used QuantiTect SYBR Green PCR Kit (Quiagen,
according
to
Colla
et
al.,
(Blood,
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Hilden, Germany). Sequences of bFGF-primers were produced by TIB Molbiol (Berlin, Germany) 2003)
using
the
following
primers:
forward:
5'-
GGCTTCTTCCTGCGCATCCAT-3'; reverse: 5'-GGTAACGGTTAGCACACACTCCTTT-3'. For internal controls, β-actin (Qiagen, Hilden, Germany) was amplified.
2.5 Enzyme-linked immunosorbent assay (ELISA)
To confirm that the ECCM-triggered stimulation of β-catenin-dependent transcription and StAR activity
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in NCI-H295R cells was mediated by bFGF, we analysed human bFGF levels in the ECCM diluted in DMEM-basal medium at increasing concentrations (Ray Biotech, Inc., Norcross, GA, USA). The plate that was coated with a specific anti-human bFGF antibodies was incubated with the following ECCM dilutions 10 %, 25 %, 50 %, 75 %, and 100 % for 2.5 hours. Subsequently, the wells were washed
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three times, treated with biotinylated antibody and then incubated with horseradish peroxidaseconjugated streptavidin, following the manufactor's protocol. Thereafter, the reaction was stopped using the provided solution, tetramethylbenzidine substrate formed a yellow color that was measured
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at 450 nm.
2.6 FGF2R immunohistochemistry Paraffin-embedded tissues of normal human adrenal glands were deparaffinized using xylene and rehydrated through a graded ethanol series. Specimens were permeabilized afterwards by incubation with 1 % Triton X-100 (Sigma Aldrich) for 5 min. After exposure to peroxidase-blocking solution (DAKO, Hamburg, Germany) for 5 min and blockade of non-specific binding sites using 10 % normal bovine serum (DAKO), tissue sections were incubated with antibodies against FGFR2 (Bec C-17, Santa Cruz Biotechnology) in a dilution of 1:100 at room temperature for 60 min. Bound immunoglobulins were detected using the Envision Detection System (DAKO). Specimens were exposed to peroxidase-labeled polymer for 30 min and afterwards with substrate chromogen solution for 10 min. Counterstaining was performed with hematoxylin II (Merck, Darmstadt, Germany) for 2 min. Washing steps were performed with D-PBS (Life technologies, Darmstadt, Germany) between all
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incubation steps. Negative control slides were incubated without primary antibody or control immunoserum.
2.7 Statistical analysis Statistical analysis was performed using ANOVA testing followed by Bonferroni’s multiple comparison post test when a Gaussian distribution was present or by Kruskal-Wallis-test with Dunns' test posthoc
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using Graph Pad Prism 4.1. Data was generated from multiple experiments with a minimum of at least 3 independent settings. If not otherwise stated, data are given as means ± SEM. P-values of 5 % were considered asstatistically relevant.
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3.Results
The present study showed that ECCM induced an increase in β-catenin-dependent transcription in
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NCI-H295R and IMAC cells. In NCI-H295R cells, the effect was dose-dependent with a stimulation 200 % over baseline at an ECCM concentration of 5 % and 700 % over baseline at an ECCM concentration of 100 %. In IMAC cells, β-catenin-dependent transcription rose over 200 % at an ECCM concentration of 75 % after 24 hours (Fig. 1). Increases were significant at a concentration of 10 % ECCM in NCI-H295R cells and at 75 % ECCM in IMACs.
Incubation of NCI-H295R cells with Wnt signaling inhibitors sFRP-1 (10 ng/mL) or Dkk-1 (1 µg/mL) in addition to 50 % ECCM did not result in a significant decrease in β-catenin-dependent transcription in
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comparison to the effect reached with 50 % ECCM alone (Fig. 2). Also, baseline β-catenin-dependent transcription was not changed by incubation with sFRP-1 or Dkk-1 (data not shown). H89, a PKA inhibitor, Akt inhibitor VIII and bisindolylmaleimide I (PKC inhibitor) did not interefere with the transcriptional activity of β-catenin (Fig. 2). Transfection experiments with a luciferase reporter
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gene under the control of multiple CRE-binding sites indicated that there is a marginal increase in cAMP-triggered activity in adrenocortical cells after being exposed to ECCM as compared to control medium. Forskolin, a known activator of cAMP-mediated transcription, served as a positive control
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(Fig. 2).
The stimulatory effect of ECCM could be significantly inhibited by U0126 (25 µM), Ly294002 (50 µM) and genistein (50 µM). The maximum decrease was in the range between 35 % and 45 % (Fig. 3). In addition, Western blot analysis showed that more β-catenin protein was present in the cytoplasm and in the nucleus of NCI-H295R cells after ECCM exposure. Translocation of β-catenin into the nucleus could be completly blocked by U0126 and Ly294002 application, indicating that MAP-kinase and the PI3-kinase pathways interfere with β-catenin-dependent transcription through β-catenin translocation (Fig. 3). ECCM stimulated β-catenin-mediated transcription to 500 % compared to untreated controls and this effect was almost completely abolished (reduction to 150 %) with Su5402, a bFGF-inhibitor (Fig. 4). Furthermore, the stimulation of NCI-H295R cells with bFGF at a concentration of 10 ng/mL led to an increase in β-catenin-dependent transcription nearly as strong as by ECCM (Fig. 4). In addition, the ECCM-induced stimulatory effect on StAR transcription (450 %) could be mimicked by bFGF in NCI-
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H295R cells to a similar degree (500 %). In line with this, Su5402 inhibited the stimulatory effect of ECCM on StAR promoter activity by more than 40 % (Fig. 4). bFGF is produced by HUVEC cells and secreted into the ECCM, as shown by the rise in concentration of bFGF in the ECCM in an ECCM-concentration-dependent manner. A maximum value of bFGF with more than 1800 pg/mL could be found in 100 % ECCM. In addition, real-time RT-PCR analysis showed bFGF mRNA levels to be 6-times higher in HUVEC as compared to NCI-H295R cells (Fig. 5).
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FGFR2 was detected ubiquitously in the cytoplasm of adrenal cortical cells by immunohistochemistry; there was no significant difference in expression of FGFR2 between the three zones of adrenal cortex (Fig.5). A minority of adrenal cortical nuclei showed an expression of FGFR2. These results confirm other studies illustrating the presence of FGFR2 in the adrenal cortex and suggesting the expression
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of FGFR1, FGFR3 and FGFR4 (Hughes SE. 1997).
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4. Discussion
Recent data support the importance of the Wnt/β-catenin pathway in adrenal gland pathophysiology (Kim et al. 2008, Schinner et al. 2009) and ECCM is known to regulate factors essential for adrenocortical development, including SF-1 and CITED2. We therefore asked the question whether ECCM regulates β-catenin activity in adrenal gland. We were able to show, that ECCM dose-dependently stimulates β-catenin-dependent transcription indicating that in addition to supplying the cells with nutrients, endothelial cells provide specific factors to adrenocortical cells for
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organ development and pathophysiology. This hypothesis is supported by data from a murine model wherein disruption of β-catenin causes disintegration of the adrenal cortex (Kim et al. 2008). Also, increased activity of β-catenin was observed in adrenal cortical tumors (Semba et al. 2000, Tissier et al. 2005, Tadjine et al. 2008, Gaujoux et al. 2008), primary pigmented nodular adrenal disease
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(Tadjine et al. 2008, Gaujoux et al. 2008) and was associated with malignancy in adrenocortical tumors (Tissier et al. 2005, Doghman et al. 2008, Berthon et al. 2010, Gaujoux et al. 2011). Of note, the NCI-H295R cell line was reported to have a mutated β-catenin with a gain in activity (Doghman et
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al. 2008). Although this effect could also be observed in our experiments, β-catenin-dependent transcription was further increased by ECCM products and was although increased in primary IMAC cells. This highlights the impact of the endothelium on adrenocortical function. Activation of β-catenin is often related to the canonical Wnt signaling pathway. Binding of Wnt molecules to frizzled receptors leads to stabilization of β-catenin and translocation into the nucleus where it binds to a Lef/Tcf-binding site (Seidensticker et al. 2000, van Amerongen et al. 2008). Adrenal cells express receptors and co-receptors sensitive to Wnt molecules (Suwa et al. 2003, Schinner et al. 2007) and are themeselves a source of Wnt glycoprotein secretion. Furthermore, Wnt molecules lead to stimulation of adrenal steroidogenesis (Heikkila et al. 2002, Gummow et al. 2003, Chen et al. 2006, Schinner et al. 2007, Kuulasmaa et al. 2008). Therefore, we studied the influence of inhibitors of Wnt signalling on ECCM-stimulated β-catenin activity. Interestingly, we found that neither sFRP nor Dkk were able to reverse the ECCM effect.
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Since the effect of ECCM on adrenal CITED2 transcription and aldosterone generation was dependent on PI3-kinase and MAP-kinase signaling (Haase et al. 2009, Ansurudeen et al. 2009), we studied the role of this pathway in the regulation of β-catenin activity by ECCM. We found that the inhibition of PI3kinase and MAP-kinase signalling prevented the ECCM-triggered increase in β-catenin activity as well as cytoplasmic and nuclear content of β-catenin protein. Similar to our findings, Yang et al., show that MEK inhibitors decrease the transcriptional activity of β-catenin in bovine retinal endothelial cells
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(Yang et al. 2010). We also found that inhibition of receptor tyrosine kinases (RTK)-inhibitors deacreased β-catenin-dependent transcription.
Basic FGF is processed by endothelial cells (Feige et al. 1989). It was shown to induce Lef/Tcfdependent transcription (Holnthoner et al. 2002), activate the MEK/ERK pathway (Yang et al. 2008) and to regulate adrenal CITED2 expression (Haase et al. 2007). Studies in fgfr-2 gene knockout mice
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demonstrate a link between FGFR-2 and adrenal cortex development (Guasti et al. 2013).
In this context, we studied whether a RTK mediating bFGF activity is involved in the stimulation of βcatenin by ECCM. Using Su5402, an inhibitor of type 3 FGF receptors, the ECCM-stimulated β-
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catenin activity and StAR promotor access could be blocked demonstrating that the type 3 FGF receptors are involved in bFGF action. In addition, incubation of adrenocortical cells with bFGF was demonstrated to mimick the effects of the ECCM on β-catenin-dependent transcription, the effect of endothelial cell products on StAR promoter activity (Ansurudeen et al. 2007, Paramonova et al., 2010) and CITED2 expression (Haase et al. 2007, Haase et al. 2009).
Our molecular and immunoassay studies confirm that endothelial cells synthesize and secrete bFGF (Hannan et al. 1988), identifying the endothelial cells as the major source of adrenal bFGF (Fig. 6)
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since bFGF was not added during the production of ECCM. However, bFGF does not seem to be the only ECCM-derived factor in stimulating the β-catenin activity because the effect of 50 % ECCM on β-catenin activity was higher than that of 1 ng/ml bFGF although the amount of bFGF in 50 % ECCM is approximately 1 ng/ml. This suggests that there are also other factors
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involved in the activation of β-catenin and not only bFGF. Thus, there may be co-factors synergizing or enhancing the bFGF-mediated stimulation. In summary, we were able to show that β-catenin activity in adrenocortical cells is regulated also by
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bFGF, secreted by endothelial cells and acting through a RTK via a pathway involving PI3/MEK kinase action. In context of the imformation of previous studies, bFGF seems to play a more prominent role for adrenal gland physiology then endothelin-1 and interleukin-6. It may even be involved in the development of the adrenal cortex or adrenocortical tumors.
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References van Amerongen R, Mikels A, Nusse R. Alternative wnt signaling is initiated by distinct receptors. Sci Signal 2008;35:re9. Ansurudeen I, Kopprasch S, Ehrhart-Bornstein M, Willenberg HS, Krug AW, Funk RHW, Bornstein SR. Vascular-adrenal niche--endothelial cell-mediated sensitization of human adrenocortical cells to angiotensin II. Horm Metab Res 2006;38:476-480.
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Ansurudeen I, Kopprasch S, Ehrhart-Bornstein M, Bornstein SR, und Willenberg HS. Endothelial cellmediated regulation of aldosterone release from human adrenocortical cells. Mol Cell Endocrinol 2007;265-266:150-156. Ansurudeen I, Willenberg HS, Kopprasch S, Krug AW, Ehrhart-Bornstein M, Bornstein SR. Endothelial factors mediate aldosterone release via PKA-independent pathways. Mol Cell Endocrinol 2009;300:66-70.
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Paramonova I, Haase M, Mülders-Opgenoorth B, Ansurudeen-Rafi I, Bornstein S R, Papewalis C, Schinner S, Schott M, Scherbaum WA, Willenberg HS. The effects of the endothelium on adrenal steroidogenesis and growth are mainly mediated by proteins other than endothelin-1. Horm Metab Res 2010;42:840-845.
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Rosolowsky LJ, Campbell WB. Endothelial cells stimulate aldosterone release from bovine adrenal zona glomerulosa cells. Am J Physiol 1994;266:E107-117. Schinner S, Willenberg HS, Krause D, Schott M, Lamounier-Zepter V, Krug A W, Ehrhart-Bornstein M, Bornstein SR, Scherbaum WA. Adipocyte-derived products induce the transcription of the StAR promoter and stimulate aldosterone and cortisol secretion from adrenocortical cells through the Wnt-signaling pathway. Int J Obes 2007;31:864-870. Schinner S, Willenberg HS, Schott M, Scherbaum WA. Pathophysiological aspects of Wnt-signaling in endocrine disease. Eur J Endocrinol 2009;160:731-737. Seidensticker MJ, Behrens J. Biochemical interactions in the wnt pathway. Biochim Biophys Acta 2000;1495:168-182. Semba S, Kusumi R, Moriya T, Sasano H. Nuclear accumulation of B-catenin in human endocrine tumors: association with Ki-67 (MIB-1) proliferative activity. Endocr Pathol 2000;11:243–250. Stratakis C A. Adrenocortical tumors, primary pigmented adrenocortical disease (PPNAD)/Carney complex, and other bilateral hyperplasias: the NIH studies. Horm Metab Res 2007;39:467-473. Suwa T, Chen M, Hawks CL, Hornsby PJ. Zonal expression of dickkopf-3 and components of the Wnt signalling pathways in the human adrenal cortex. J Endocrinol 2003;178:149-158.
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Vane JR, Botting RM. Formation by the endothelium of prostacyclin, nitric oxide and endothelin. J Lipid Mediat 1993;6:395-404. Vinson GP, Pudney JA, Whitehouse BJ. The mammalian adrenal circulation and the relationship between adrenal blood flow and steroidogenesis. J Endocrinol 1985;105:285-294.
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Werminghaus P, Haase M, Hornsby PE, Schinner S, Schott M, Lammer B, Goretzki PE, Scherbaum WA, Willenberg HS. Studies on the role of hedgehog signaling in normal human adrenal gland and adrenal tumors. J Steroid Biochem Mol Biol 2014;139:7–15. Willenberg HS, Ansurudeen I, Schebesta K, Haase M, Wess B, Schinner S, Raffel A, Schott M, Scherbaum WA. The endothelium secretes interleukin-6 (IL-6) and induces IL-6 and aldosterone generation by adrenocortical cells. Exp Clin Endocrinol Diabetes 2008;116:S70-74. Willenberg HS, Schinner S, Ansurudeen I. New mechanisms to control aldosterone synthesis. Horm Metab Res 2008;40:435-441.
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Yang H, Xia Y, Lu S Q, Soong T W, Feng Z W. Basic fibroblast growth factor-induced neuronal differentiation of mouse bone marrow stromal cells requires FGFR-1, MAPK/ERK, and transcription cator AP-1. J Biol Chem 2008;283:5287-95. Yang J, Duh EJ, Caldwell RB, Behzadian MA. Antipermeability function of PEDF involves blockade of the MAP kinase/GSK/beta-catenin signaling pathway and uPAR expression. Invest Ophthalmol Vis Sci 2010;51:3273-3280.
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Zhang DX, Gauthier KM, Falck JR, Siddam A, Campbell WB. Steroid-producing cells regulate arterial tone of adrenal cortical arteries. Endocrinology 2007;148:3569-3576.
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Figure 1 Panel A: Endothelial cell conditioned medium (ECCM) stimulates beta-catenin dependent transcription in NCI-H295R cells. NCI-H295R cells were incubated for 24 hours with increasing concentrations of ECCM (5 %, 10 %, 20 %, 30 %, 50 %, 75 %, 100 %). Panel B: Endothelial cell conditioned medium (ECCM) also stimulates beta-catenin dependent transcription in immortalized adrenocortical cells.
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Immortalized adrenocortical cells were incubated for 24 hours with increasing concentrations of ECCM
Panel A
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(25 %, 50 %, 75 %, 100 %).
Panel B
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Figure 2 Wnt signaling- and protein kinase pathways inhibitors are not able to block the stimulatory effect of endothelial cell conditioned medium (ECCM) on beta-catenin dependent transcription in NCI-H295R cells. Fig. 2, Panel A: NCI-H295R cells were treated with 50 % ECCM and Wnt inhibitors, including soluble frizzled protein (sFRP-1, 10 ng/ml) or dickkopf protein (Dkk-1, 1 µg/ml). Fig. 2, Panel B:
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Incubation of NCI-H295 R cells with 50 % ECCM, H89 (10 µM), Akt inhibitor VIII (Akt I-VIII, 1 µM), or bisindolylmaleimide I (BIM I, 3 µM). Fig. 2, Panel C: Transfection experiments with a luciferase reporter gene under the control of multiple CRE-binding sites indicated that there is a marginal
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increase in cAMP-triggered activity in adrenocortical cells after being exposed to ECCM as compared to control medium. Forskolin, a known activator of cAMP-mediated transcription, served as a positive
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control.
Panel A
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Panel B
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Panel C
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Figure 3 The endothelial cell -mediated stimulation of beta-catenin in NCI-H295R cells is reversed by compounds inhibiting mitogen-activated protein kinase (MEK), phosphatidylinositol 3- (PI3K) and receptor tyrosine (RTK) kinases. Fig. 3, Panel A: ECCM-induced stimulation of beta-catenindependent transcription is inhibited by MEK, phosphatidylinositol 3- (PI3-) kinase RTK inhibitors. NCI-
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H295R cells were treated for 24 hours with 50 % ECCM in presence of the inhibitors U0126 (25 µM), Ly294002 (50 µM), and genistein (50 µM). Fig. 3, Panel B: Beta-catenin activity in the cytoplasm and translocation to the nucleus can be inhibited by MEK and PI3-kinase inhibitors. NCI-H295R cells were
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incubated with 50% endothelial cell conditioned medium (ECCM) in presence or absence of U0126 (25 µM) and Ly294002 (50 µM) for 24 hours. The lysates were analyzed with the indicated antibody of
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beta-catenin.
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Panel A
Panel B
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Figure 4 Relative luciferase activity in NCI-H295R cells after exposure to ECCM with or without SU5402 (22 µM) and to bFGF at increasing concentrations (1 ng/ml, 10 ng/ml) for 24 hours. Fig.4, Panel A: The stimulation of beta-catenin-dependent transcription through endothelial cell conditioned medium (ECCM) can be mimicked by basic firoblast growth factor (bFGF) and inhibited by bFGF inhibitor
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(Panel A). Fig.4, Panel B: The stimulation of steroidogenic acute regulatory protein (StAR) activity through endothelial cell conditioned medium (ECCM) can be mimicked by basic firoblast growth factor
Panel A
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(bFGF) and inhibited by bFGF inhibitor.
Panel B
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Figure 5 Panel A: Enzyme Linked Immunosorbent Assay (ELISA) analysis show that rising concentrations of endothelial cell conditioned medium (ECCM) show increasing concentrations of basic fibroblast growth factor (bFGF). Panel B: Basic fibroblast growth factor (bFGF) mRNA expression in NCI-H295R cells vs. in human umbilical vein endothelial cells (HUVEC). Panels C and D: Representative section of a
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normal human adrenal gland displaying immunoractivity to an antibody against the type 2 fibroblast
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growth factor receptor (C) and a negative control section (D).
Panel B
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Panel A
Panel C
Panel D
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Figure 6 This scheme summarizes the findings of this paper and that of Haase et al., (Haase et al., J Endocrinol 2007). In addition to other factors, endothelial cells synthesize and secrete basic fibroblast growth factor (bFGF) that binds to its receptor on adrenocortical cells and promotes β-catenin-dependent transcription. Corticotropin (ACTH) and angiotensin II (ATII) are
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physiologic regulators of adrenal steroidogenesis and bind to adrenal ACTH (MC2R) or ATII
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type 1 (AT1R) receptors, respectively.
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Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
1
1
1
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Short Title:
1
1
Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
2
5
1,2
,
2,3
, Peter J.
1
Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S. 1,6
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Willenberg
Highlights
endothelial cells induce β-catenin activity in adrenal cortical cells
•
Wnt-signalling, cAMP, Akt or PKC pathways are not involved
•
MEK, PI3-kinase and receptor tyrosine kinases are involved
•
stimulation of β-catenin and StAR activity is partly mimicked by bFGF
•
bFGF is produced and secreted by human umbilical cord endothelial cells
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•
S0303-7207(16)30484-1
DOI:
10.1016/j.mce.2016.11.015
Reference:
MCE 9729
To appear in:
Molecular and Cellular Endocrinology
Received Date: 20 June 2016 Revised Date:
17 November 2016
Accepted Date: 20 November 2016
Please cite this article as: Schwafertz, C., Schinner, S., Kühn, M.C., Haase, M., Asmus, A., MüldersOpgenoorth, B., Ansurudeen, I., Hornsby, P.J., Morawietz, H., Oetjen, E., Schott, M., Willenberg, H.S., Endothelial cells regulate β-catenin activity in adrenocortical cells via secretion of basic fibroblast growth factor, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.11.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Schwafertz / Willenberg et al.,
β-catenin and bFGF – graphical abstract
ACCEPTED MANUSCRIPT
Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
1
1
1
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Short Title:
1
1
Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
2
5
1,2
,
2,3
, Peter J.
1
Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S. 1,6
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Willenberg
Graphical Abstract
Endothelial cell products are capable in regulating SF-1, StAR and CITED2, essential factor for adrenal organogenesis or function. However, the signalling involved was net yet conclusively described. Schwafertz et al. have found that β-catenin-mediated transcription is involved in the process and that endothelial cell products do not use the classical wnt-signalling pathway. The secretion of bFGF by endothelial cells partly explains the effects.
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Fig. 6
Schwafertz / Willenberg et al.,
β-catenin and bFGF – Title page
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Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
1
1
RI PT
Short Title:
1
1
1
Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
2
5
2,3
1,2
,
, Peter J.
1
Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S.
Affiliations:
1
1,6
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Willenberg
Division for Specific Endocrinology, Medical Faculty, Heinrich-Heine
University Dusseldorf; D-40225 Dusseldorf;
2
Department of Medicine III, Carl
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Gustav Carus Medical School, University of Technology, D-01307 Dresden, 3 Germany; Department of Molecular Medicine and Surgery, L1:01 Rolf Luft 4
Centrum, Karolinska Institute, Stockholm, Sweden; Department of Physiology and Sam and Ann Barshop Institute for Longevity and Aging 5
Studies, University of Texas Health Science Center, San Antonio, TX, USA; 6 Institute of Pharmacology, University of Göttingen; Germany; Division of Endocrinology and Metabolism, Rostock University Medical Center, Germany Holger S. Willenberg
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Correspondence to:
Division of Endocrinology and Metabolism Rostock University Medical Center Ernst-Heydemann-Str, 6, D-18057 Rostock, Germany
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Tel: +49 (381) 494-7521; Fax: +49 (381) 494-7522 eMail: [email protected]
The plasmid for StAR Promoter was a generous gift from Dr. D. Stocco
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Acknowledgement:
(Texas Tech University Health Sciences Center, Lubbock, USA). This work was supported by the Doktor Robert Pfleger-Stiftung, Bamberg, Germany (HSW).
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Abstract
Endothelial cell-derived products influence the synthesis of aldosterone and cortisol in human adrenocortical cells by modulating proteins such as steroidogenic acute-regulatory (StAR) protein, steroidogenic factor (SF)-1 and CITED2. However, the potential endothelial cell-derived factors that mediate this effect are still unknown. The current study was perfomed to look into the control of β-
catenin activity in adrenocortical NCI-H295R cells.
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catenin activity by endothelial cell-derived factors and to identify a mechanism by which they affect β-
Using reporter gene assays and Western blotting, we found that endothelial cell-conditioned medium (ECCM) led to nuclear translocation of β-catenin and an increase in β-catenin-dependent transcription that could be blocked by U0126, an inhibitor of the mitogen-activated protein kinase pathway.
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Furthermore, we found that a receptor tyrosin kinase (RTK) was involved in ECCM-induced β-catenindependent transcription. Through selective inhibition of RTK using Su5402, it was shown that receptors responding to basic fibroblast growth factor (bFGF) mediate the action of ECCM.
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Adrenocortical cells treated with bFGF showed a significant greater level of bFGF mRNA. In addition, HUVECs secrete bFGF in a density-dependent manner. In conclusion, the data suggest that endothelial cells regulate β-catenin activity in adrenocortical cells also via secretion of basic fibroblast growth factor.
aldosterone; adrenal gland; beta-catenin; basic fibroblast growth factor; CITED2
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Keywords:
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1. Introduction
The adrenal gland is an effector organ of the hypothalamo-pituitary-adrenal axis and the reninangiotensin system. As such, it is involved in the regulation of salt and water homeostasis, extracellular volume balance, blood pressure, immune function, and energy metabolism in response to stress.
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The adrenal gland is heavily vascularized and almost every adrenocortical cell is in direct contact to an endothelial cell, whereby the microstructure is of relevance for proper adrenal function (Dobbie et al. 1966, Hornsby et al. 1987, Ehrhart-Bornstein et al. 1998). The regulation of adrenal blood flow is dependent on corticotropin (ACTH) (Vinson et al. 1985, Breslow et al. 1992, Thomas et al. 2003, Mohn et al. 2005, Zhang et al. 2007) and endothelial cell products affect the synthesis of adrenocortical
Ehrhart-Bornstein et al. 1998, Willenberg et al. 2008).
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hormones, including aldosterone (Vinson et al. 1985, Hinson et al. 1991, Nussdorfer et al. 1997,
Although a number of specific endothelial cell-derived factors have been identified, the overall effect of
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endothelial cell-derived products on adrenocortical cells has been poorly studied. Endothelial cellconditioned medium (ECCM) is a cocktail of factors, containing endothelin-1, angiotensin-2 (Kifor et al. 1987, Vane et al. 1993), interleukin-6 (Willenberg et al. 2008) and other, unidentified proteins (EhrhartBornstein et al. 1998, Willenberg et al. 2008). The aforementioned known components of the ECCM do not explain the effects observed when adrenocortical cells were incubated with ECCM (Ansurudeen et al. 2006, Ansurudeen et al. 2007, Willenberg et al. 2008, Haase et al. 2009, Ansurudeen et al. 2009, Paramonova et al. 2010). However, Rosolowski et al., described an endothelium-derived 3000
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Da protein which leads to increased aldosterone concentrations in supernatants of cultured bovine adrenal cells via the protein kinase C pathway (Rosolowsky et al. 1994). Interestingly, our studies have shown that the endothelial cells act on adrenocortical cells involving proteins essential for adrenocortical development, including steroidogenic factor (SF)-1, steroidogenic
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acute regulatory protein (StAR) and CITED2 (Ansurudeen et al. 2007, Haase et al. 2009). In addition, endothelial cell-derived products influence the proliferation of adrenocortical cells (Paramonova et al. 2010).
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Recently, proliferation and tumorigenesis of adrenocortical adenomas were demonstrated to be associated with activation of β-catenin (Tissier et al. 2005, Stratakis et al. 2007, Schinner et al. 2009, Berthon et al. 2010, Gaujoux et al. 2011). Furthermore, knock-down of β-catenin leads to a decrease in SF-1 positive cells as well as to an early disappearance of adrenocortical and adrenomedullary precursor cells (Kim et al. 2008). It was shown by other groups and ours that adrenocortical cells express frizzled receptors and βcatenin in the human adrenal gland and that activation of the Wnt signalling pathway stimulates aldosterone and cortisol biosynthesis through β-catenin (Chen et al. 2006, Schinner et al. 2007, Tadjine et al. 2008, Schinner et al. 2009). We therefore hypothesized whether ECCM could regulate βcatenin-dependent transcription and if so whether the Wnt signalling pathway is involved in its regulation. We here show that ECCM significantly up-regulates β-catenin-dependent transcription through MAP- and RTK-pathways.
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2. Materials and methods
This study aimed at better characterization of the ECCM effect on adrenocortical cells that was seen in previous studies whereby data on the adrenal hormonal responses and adrenal cell viability have been made available to the public (Ansurudeen et al. 2006, Ansurudeen et al. 2007, Ansurudeen et al.
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2009, Haase et al. 2009, Paramonova et al. 2010). Further experiments were conducted as follows.
2.1 Cell cultures
For our in vitro studies, three cell lines were used. NCI-H295R cells were cultured in DMEM/F12Glutamax medium (Invitrogen, Karlsruhe, Germany), containing fetal bovine serum (2 %, Invitrogen),
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insulin (66 nM, Sigma-Aldrich, Munich, Germany), hydrocortisone (10 nM, Sigma-Aldrich), apotransferrin (10 µg/mL, Sigma-Aldrich), β-estradiol (10 nM, Sigma-Aldrich), sodium selenite (30 nM, Sigma-Aldrich), penicillin (100 U/mL, Invitrogen) and streptomycin (100 mg/mL, Invitrogen), at 37°C in
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a humidified atmosphere of 95 % air, and 5 % CO2, as described previously (Ansurudeen et al. 2007). Primary adrenocortical cells were established from an adrenal gland of a patient who had undergone unilateral nephrectomy for renal cancer. An immortalized as described previously (Suwa et al. 2001), using pLEGFP-CMV-SV40T
and pBabe-hTERT
retroviral vectors to produce immortalized
adrenocortical cells. Immortalized adrenocortical (IMAC) cells were cultured in DMEM/F12-Glutamax (Invitrogen) with 10 % fetal bovine serum (Invitrogen), penicillin (100 U/ml, Invitrogen) and streptomycin (100 mg/ml, Invitrogen) at 37°C in a humidified atmosphere of 95 % air, and 5 % CO2 for
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further experiments (Werminghaus et al. 2014).
At confluency, H295R and IMAC cells were subcultured using Accutase (PAA Laboratories, Cölbe, 6
2
Germany) and seeded at density of 2×10 cells in 75-cm flasks. For transfection and protein extraction, cells were plated in 24-well culture plates. At confluency of 80%, cells were used for
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experimental procedures.
Different preparations of human umbilical vein endothelial cells (HUVEC) were used and obtained from normal term and normal pregnancy donors. HUVECs have been isolated using the following
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protocol (Ansurudeen et al., 2007). After being emptied of blood by perfusion with phosphate buffer (50 mM, pH 7.0), 15-cm-long segments of umbilical cord venous or arterial vessels were filled with collagenase I (1 mg/mL of endothelial cell medium, PromoCell, Heidelberg, Germany) and incubated at 37°C for 30 min. Cells were collected from the suspension by centrifugation at 50 g for 5 min at 4°C. The cell pellet was suspended and cultured in endothelial cell basal medium (PromoCell), containing the ingredients of the supplement pack (PromoCell), fetal calf serum (10 %) and antibiotics (penicillin 100 U/mL and streptomycin 100 µg/mL), at 37°C in a humidified atmosphere of 95 % air and 5% CO2. For experiments, cells from passages 3 – 5 were used. Commercially available HUVEC and HUAEC preparations (Lonza, Walkersville, USA) were also used. At a confluency of over 80 %, fresh DMEM/F12 glutamax medium with the supplemental pack was added to endothelial cells and incubated for 48-72 hours to obtain ECCM. After this incubation period, ECCM was centrifuged to remove potential contaminat cells and stored at –20°C until further use.
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2.2 Plasmids and transfections The plasmid TOPFLASH reporter gene (with multiple binding sites for TCF/LEF-transcription factors which are co-activated by β-catenin) and the FOPFLASH reporter gene (with mutated TCF/LEFbinding-sites) are commercially available at UPSTATE biotechnology (Lake Placid, USA). The plasmids pT81 for studies of cAMP-dependent protein kinase A transcription as well as the 1300 StAR-Luc plasmid were used as described previously (Oetjen et al. 1994, Caron et al. 1997). Cells 5
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were seeded at a density of 2×10 cells per well in 24-well culture plates. After 24 h, when confluency exceeded 50 %, transfection was performed with Nanofectamin (PAA Laboratories, Cölbe, Germany) according to the manufacturer's protocol. The plasmids TOPFLASH, FOPFLASH, StAR-Luc and pT81 plasmids were transfected at a concentration of 0.5 µg per well along with the renilla luciferase pRLTK-Luc plasmid (Promega, Mannheim, Germany) at a concentration of 0.1 µg for internal control.
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24 h later, the cells were stimulated with DMEM/F12 basal medium or ECCM, alongwith various inhibitors or proteins for next 24 h. The NCI-H295R cells and the immortalized cells were treated with different concentrations of ECCM diluted in DMEM/F12 (5 %, 10 %, 20 %, 25 %, 30 %, 50 %, 75 %,
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and 100 %). The effect of Wnt signaling in NCI-H295R cells was studied in the presence of inhibitors of the Wnt signaling pathway using sFRP-1 (secreted frizzled-related protein-1; 10 ng/mL, R&D Systems, Inc., Minneapolis, USA) which interacts with Wnt ligands and impedes their binding to one of the frizzled receptors, or Dkk-1 (Dickkopf-related protein 1; 1 µg/mL, R&D Systems, Inc.) which binds to the LRP5/6 co-receptor and inhibits signal transduction. The role of protein kinases in NCI-H295R cells were studied using inhibitors of protein kinase A (H89, 10 µM, Calbiochem Merck, Darmstadt, Germany), protein kinase B (Akt inhibitor VIII, 1 µM, Calbiochem Merck), protein kinase C
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(bisindolylmaleimide I, 3 µM, Calbiochem Merck), MEK inhibitor (U0126, 25 µM, Promega), PI3-kinase inhibitor (Ly294002, 50 µM, Sigma-Aldrich) and genistein (inhibitor of tyrosine-specific protein kinases, 50 µM, Sigma-Aldrich). Briefly, NCI-H295R cells were incubated with 50 % ECCM with or without inhibitors and studied. Other stimulation experiments in NCI-H295R cells were performed with 50 %
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ECCM, including the bFGF receptor inhibitor Su5402 (22 µM, Merck) or bFGF (PromoCell, Heidelberg, Germany) at concentrations of 1 ng/mL or 10 ng/mL. Transfection experiments were also carried out with the StAR reporter gene in H295R cells, treated with 10 %, 25 % or 50 % ECCM as
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described previously (Paramonova et al. 2010). After the stimulation period, analyses were performed with the dual-luciferase reporter assay system (Promega) as described previously (Haase et al. 2009). Results are expressed as means ± standard deviation (SD) of a minimum of three independent experiments. Statistical analysis was performed with a t-test using Prism 4.1 (Graph Pad, San Diego, CA, USA).
2.3 Nuclear extraction and Western blot NCI-H295R cells were seeded in 6-well culture plates in DMEM/F12 medium. Cells were treated with 50% ECCM and U0126 (25 µM) or Ly294002 (50 µM) for 24h. For total protein extraction cells were lysed with lysis buffer 1 (10 mM HEPES pH 7.9; 10 mM KCl; 0.1 mM EDTA) containing 1 % Nonidet P40. For nuclear extraction, cells were treated first with lysis buffer 1 and then with lysis buffer 2 (20 mM HEPES pH 7.9; 0.4 M NaCl; 1 mM EDTA). Concentrations of the extracted proteins were determined using the BCA Protein Assay kit (Thermo Scientific, Rockford, IL, USA).
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Denatured proteins were resolved by SDS-polyacrylamide gel electrophoresis, followed by transferring the proteins onto a nitrocellulose membrane. The blots were incubated with specific rabbit anti-β-catenin antibody (Cell Signaling Technology, Danvers, MA, USA) at 1:1000 dilution, bovine secondary antibody (Dako Cytomation, Glostrup, Denmark) at a 1:5000 dilution and anti-β-actin antibody (Sigma-Aldrich, St. Louis, MO, USA) at a 1:1000 dilution for normalization. Protein detection was performed with chemiluminescence using ECL
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Plus Western blotting detection reagents (GE Healthcare, Buckinghamshire, UK) after incubation with HRP-Streptavidin (Jackson ImmunoResearch Laboratories, Suffolk, UK).
2.4 Reverse transcriptase-PCR (RT-PCR)
RNA was extracted from NCI-H295R cells and HUVEC using RNeasy MiniKit (Qiagen, Hilden,
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Germany) following the manufactor's instructions (Haase et al. 2009). Synthesis of cDNA was performed using the Ready-to-go t-primed first-strand kit (GE Healthcare, Buckinghamshire, UK). For amplification and detection of PCR products we used QuantiTect SYBR Green PCR Kit (Quiagen,
according
to
Colla
et
al.,
(Blood,
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Hilden, Germany). Sequences of bFGF-primers were produced by TIB Molbiol (Berlin, Germany) 2003)
using
the
following
primers:
forward:
5'-
GGCTTCTTCCTGCGCATCCAT-3'; reverse: 5'-GGTAACGGTTAGCACACACTCCTTT-3'. For internal controls, β-actin (Qiagen, Hilden, Germany) was amplified.
2.5 Enzyme-linked immunosorbent assay (ELISA)
To confirm that the ECCM-triggered stimulation of β-catenin-dependent transcription and StAR activity
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in NCI-H295R cells was mediated by bFGF, we analysed human bFGF levels in the ECCM diluted in DMEM-basal medium at increasing concentrations (Ray Biotech, Inc., Norcross, GA, USA). The plate that was coated with a specific anti-human bFGF antibodies was incubated with the following ECCM dilutions 10 %, 25 %, 50 %, 75 %, and 100 % for 2.5 hours. Subsequently, the wells were washed
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three times, treated with biotinylated antibody and then incubated with horseradish peroxidaseconjugated streptavidin, following the manufactor's protocol. Thereafter, the reaction was stopped using the provided solution, tetramethylbenzidine substrate formed a yellow color that was measured
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at 450 nm.
2.6 FGF2R immunohistochemistry Paraffin-embedded tissues of normal human adrenal glands were deparaffinized using xylene and rehydrated through a graded ethanol series. Specimens were permeabilized afterwards by incubation with 1 % Triton X-100 (Sigma Aldrich) for 5 min. After exposure to peroxidase-blocking solution (DAKO, Hamburg, Germany) for 5 min and blockade of non-specific binding sites using 10 % normal bovine serum (DAKO), tissue sections were incubated with antibodies against FGFR2 (Bec C-17, Santa Cruz Biotechnology) in a dilution of 1:100 at room temperature for 60 min. Bound immunoglobulins were detected using the Envision Detection System (DAKO). Specimens were exposed to peroxidase-labeled polymer for 30 min and afterwards with substrate chromogen solution for 10 min. Counterstaining was performed with hematoxylin II (Merck, Darmstadt, Germany) for 2 min. Washing steps were performed with D-PBS (Life technologies, Darmstadt, Germany) between all
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incubation steps. Negative control slides were incubated without primary antibody or control immunoserum.
2.7 Statistical analysis Statistical analysis was performed using ANOVA testing followed by Bonferroni’s multiple comparison post test when a Gaussian distribution was present or by Kruskal-Wallis-test with Dunns' test posthoc
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using Graph Pad Prism 4.1. Data was generated from multiple experiments with a minimum of at least 3 independent settings. If not otherwise stated, data are given as means ± SEM. P-values of 5 % were considered asstatistically relevant.
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3.Results
The present study showed that ECCM induced an increase in β-catenin-dependent transcription in
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NCI-H295R and IMAC cells. In NCI-H295R cells, the effect was dose-dependent with a stimulation 200 % over baseline at an ECCM concentration of 5 % and 700 % over baseline at an ECCM concentration of 100 %. In IMAC cells, β-catenin-dependent transcription rose over 200 % at an ECCM concentration of 75 % after 24 hours (Fig. 1). Increases were significant at a concentration of 10 % ECCM in NCI-H295R cells and at 75 % ECCM in IMACs.
Incubation of NCI-H295R cells with Wnt signaling inhibitors sFRP-1 (10 ng/mL) or Dkk-1 (1 µg/mL) in addition to 50 % ECCM did not result in a significant decrease in β-catenin-dependent transcription in
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comparison to the effect reached with 50 % ECCM alone (Fig. 2). Also, baseline β-catenin-dependent transcription was not changed by incubation with sFRP-1 or Dkk-1 (data not shown). H89, a PKA inhibitor, Akt inhibitor VIII and bisindolylmaleimide I (PKC inhibitor) did not interefere with the transcriptional activity of β-catenin (Fig. 2). Transfection experiments with a luciferase reporter
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gene under the control of multiple CRE-binding sites indicated that there is a marginal increase in cAMP-triggered activity in adrenocortical cells after being exposed to ECCM as compared to control medium. Forskolin, a known activator of cAMP-mediated transcription, served as a positive control
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(Fig. 2).
The stimulatory effect of ECCM could be significantly inhibited by U0126 (25 µM), Ly294002 (50 µM) and genistein (50 µM). The maximum decrease was in the range between 35 % and 45 % (Fig. 3). In addition, Western blot analysis showed that more β-catenin protein was present in the cytoplasm and in the nucleus of NCI-H295R cells after ECCM exposure. Translocation of β-catenin into the nucleus could be completly blocked by U0126 and Ly294002 application, indicating that MAP-kinase and the PI3-kinase pathways interfere with β-catenin-dependent transcription through β-catenin translocation (Fig. 3). ECCM stimulated β-catenin-mediated transcription to 500 % compared to untreated controls and this effect was almost completely abolished (reduction to 150 %) with Su5402, a bFGF-inhibitor (Fig. 4). Furthermore, the stimulation of NCI-H295R cells with bFGF at a concentration of 10 ng/mL led to an increase in β-catenin-dependent transcription nearly as strong as by ECCM (Fig. 4). In addition, the ECCM-induced stimulatory effect on StAR transcription (450 %) could be mimicked by bFGF in NCI-
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H295R cells to a similar degree (500 %). In line with this, Su5402 inhibited the stimulatory effect of ECCM on StAR promoter activity by more than 40 % (Fig. 4). bFGF is produced by HUVEC cells and secreted into the ECCM, as shown by the rise in concentration of bFGF in the ECCM in an ECCM-concentration-dependent manner. A maximum value of bFGF with more than 1800 pg/mL could be found in 100 % ECCM. In addition, real-time RT-PCR analysis showed bFGF mRNA levels to be 6-times higher in HUVEC as compared to NCI-H295R cells (Fig. 5).
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FGFR2 was detected ubiquitously in the cytoplasm of adrenal cortical cells by immunohistochemistry; there was no significant difference in expression of FGFR2 between the three zones of adrenal cortex (Fig.5). A minority of adrenal cortical nuclei showed an expression of FGFR2. These results confirm other studies illustrating the presence of FGFR2 in the adrenal cortex and suggesting the expression
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of FGFR1, FGFR3 and FGFR4 (Hughes SE. 1997).
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4. Discussion
Recent data support the importance of the Wnt/β-catenin pathway in adrenal gland pathophysiology (Kim et al. 2008, Schinner et al. 2009) and ECCM is known to regulate factors essential for adrenocortical development, including SF-1 and CITED2. We therefore asked the question whether ECCM regulates β-catenin activity in adrenal gland. We were able to show, that ECCM dose-dependently stimulates β-catenin-dependent transcription indicating that in addition to supplying the cells with nutrients, endothelial cells provide specific factors to adrenocortical cells for
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organ development and pathophysiology. This hypothesis is supported by data from a murine model wherein disruption of β-catenin causes disintegration of the adrenal cortex (Kim et al. 2008). Also, increased activity of β-catenin was observed in adrenal cortical tumors (Semba et al. 2000, Tissier et al. 2005, Tadjine et al. 2008, Gaujoux et al. 2008), primary pigmented nodular adrenal disease
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(Tadjine et al. 2008, Gaujoux et al. 2008) and was associated with malignancy in adrenocortical tumors (Tissier et al. 2005, Doghman et al. 2008, Berthon et al. 2010, Gaujoux et al. 2011). Of note, the NCI-H295R cell line was reported to have a mutated β-catenin with a gain in activity (Doghman et
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al. 2008). Although this effect could also be observed in our experiments, β-catenin-dependent transcription was further increased by ECCM products and was although increased in primary IMAC cells. This highlights the impact of the endothelium on adrenocortical function. Activation of β-catenin is often related to the canonical Wnt signaling pathway. Binding of Wnt molecules to frizzled receptors leads to stabilization of β-catenin and translocation into the nucleus where it binds to a Lef/Tcf-binding site (Seidensticker et al. 2000, van Amerongen et al. 2008). Adrenal cells express receptors and co-receptors sensitive to Wnt molecules (Suwa et al. 2003, Schinner et al. 2007) and are themeselves a source of Wnt glycoprotein secretion. Furthermore, Wnt molecules lead to stimulation of adrenal steroidogenesis (Heikkila et al. 2002, Gummow et al. 2003, Chen et al. 2006, Schinner et al. 2007, Kuulasmaa et al. 2008). Therefore, we studied the influence of inhibitors of Wnt signalling on ECCM-stimulated β-catenin activity. Interestingly, we found that neither sFRP nor Dkk were able to reverse the ECCM effect.
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Since the effect of ECCM on adrenal CITED2 transcription and aldosterone generation was dependent on PI3-kinase and MAP-kinase signaling (Haase et al. 2009, Ansurudeen et al. 2009), we studied the role of this pathway in the regulation of β-catenin activity by ECCM. We found that the inhibition of PI3kinase and MAP-kinase signalling prevented the ECCM-triggered increase in β-catenin activity as well as cytoplasmic and nuclear content of β-catenin protein. Similar to our findings, Yang et al., show that MEK inhibitors decrease the transcriptional activity of β-catenin in bovine retinal endothelial cells
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(Yang et al. 2010). We also found that inhibition of receptor tyrosine kinases (RTK)-inhibitors deacreased β-catenin-dependent transcription.
Basic FGF is processed by endothelial cells (Feige et al. 1989). It was shown to induce Lef/Tcfdependent transcription (Holnthoner et al. 2002), activate the MEK/ERK pathway (Yang et al. 2008) and to regulate adrenal CITED2 expression (Haase et al. 2007). Studies in fgfr-2 gene knockout mice
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demonstrate a link between FGFR-2 and adrenal cortex development (Guasti et al. 2013).
In this context, we studied whether a RTK mediating bFGF activity is involved in the stimulation of βcatenin by ECCM. Using Su5402, an inhibitor of type 3 FGF receptors, the ECCM-stimulated β-
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catenin activity and StAR promotor access could be blocked demonstrating that the type 3 FGF receptors are involved in bFGF action. In addition, incubation of adrenocortical cells with bFGF was demonstrated to mimick the effects of the ECCM on β-catenin-dependent transcription, the effect of endothelial cell products on StAR promoter activity (Ansurudeen et al. 2007, Paramonova et al., 2010) and CITED2 expression (Haase et al. 2007, Haase et al. 2009).
Our molecular and immunoassay studies confirm that endothelial cells synthesize and secrete bFGF (Hannan et al. 1988), identifying the endothelial cells as the major source of adrenal bFGF (Fig. 6)
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since bFGF was not added during the production of ECCM. However, bFGF does not seem to be the only ECCM-derived factor in stimulating the β-catenin activity because the effect of 50 % ECCM on β-catenin activity was higher than that of 1 ng/ml bFGF although the amount of bFGF in 50 % ECCM is approximately 1 ng/ml. This suggests that there are also other factors
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involved in the activation of β-catenin and not only bFGF. Thus, there may be co-factors synergizing or enhancing the bFGF-mediated stimulation. In summary, we were able to show that β-catenin activity in adrenocortical cells is regulated also by
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bFGF, secreted by endothelial cells and acting through a RTK via a pathway involving PI3/MEK kinase action. In context of the imformation of previous studies, bFGF seems to play a more prominent role for adrenal gland physiology then endothelin-1 and interleukin-6. It may even be involved in the development of the adrenal cortex or adrenocortical tumors.
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Figure 1 Panel A: Endothelial cell conditioned medium (ECCM) stimulates beta-catenin dependent transcription in NCI-H295R cells. NCI-H295R cells were incubated for 24 hours with increasing concentrations of ECCM (5 %, 10 %, 20 %, 30 %, 50 %, 75 %, 100 %). Panel B: Endothelial cell conditioned medium (ECCM) also stimulates beta-catenin dependent transcription in immortalized adrenocortical cells.
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Immortalized adrenocortical cells were incubated for 24 hours with increasing concentrations of ECCM
Panel A
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(25 %, 50 %, 75 %, 100 %).
Panel B
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Figure 2 Wnt signaling- and protein kinase pathways inhibitors are not able to block the stimulatory effect of endothelial cell conditioned medium (ECCM) on beta-catenin dependent transcription in NCI-H295R cells. Fig. 2, Panel A: NCI-H295R cells were treated with 50 % ECCM and Wnt inhibitors, including soluble frizzled protein (sFRP-1, 10 ng/ml) or dickkopf protein (Dkk-1, 1 µg/ml). Fig. 2, Panel B:
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Incubation of NCI-H295 R cells with 50 % ECCM, H89 (10 µM), Akt inhibitor VIII (Akt I-VIII, 1 µM), or bisindolylmaleimide I (BIM I, 3 µM). Fig. 2, Panel C: Transfection experiments with a luciferase reporter gene under the control of multiple CRE-binding sites indicated that there is a marginal
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increase in cAMP-triggered activity in adrenocortical cells after being exposed to ECCM as compared to control medium. Forskolin, a known activator of cAMP-mediated transcription, served as a positive
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Figure 3 The endothelial cell -mediated stimulation of beta-catenin in NCI-H295R cells is reversed by compounds inhibiting mitogen-activated protein kinase (MEK), phosphatidylinositol 3- (PI3K) and receptor tyrosine (RTK) kinases. Fig. 3, Panel A: ECCM-induced stimulation of beta-catenindependent transcription is inhibited by MEK, phosphatidylinositol 3- (PI3-) kinase RTK inhibitors. NCI-
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H295R cells were treated for 24 hours with 50 % ECCM in presence of the inhibitors U0126 (25 µM), Ly294002 (50 µM), and genistein (50 µM). Fig. 3, Panel B: Beta-catenin activity in the cytoplasm and translocation to the nucleus can be inhibited by MEK and PI3-kinase inhibitors. NCI-H295R cells were
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incubated with 50% endothelial cell conditioned medium (ECCM) in presence or absence of U0126 (25 µM) and Ly294002 (50 µM) for 24 hours. The lysates were analyzed with the indicated antibody of
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Figure 4 Relative luciferase activity in NCI-H295R cells after exposure to ECCM with or without SU5402 (22 µM) and to bFGF at increasing concentrations (1 ng/ml, 10 ng/ml) for 24 hours. Fig.4, Panel A: The stimulation of beta-catenin-dependent transcription through endothelial cell conditioned medium (ECCM) can be mimicked by basic firoblast growth factor (bFGF) and inhibited by bFGF inhibitor
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(Panel A). Fig.4, Panel B: The stimulation of steroidogenic acute regulatory protein (StAR) activity through endothelial cell conditioned medium (ECCM) can be mimicked by basic firoblast growth factor
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(bFGF) and inhibited by bFGF inhibitor.
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Figure 5 Panel A: Enzyme Linked Immunosorbent Assay (ELISA) analysis show that rising concentrations of endothelial cell conditioned medium (ECCM) show increasing concentrations of basic fibroblast growth factor (bFGF). Panel B: Basic fibroblast growth factor (bFGF) mRNA expression in NCI-H295R cells vs. in human umbilical vein endothelial cells (HUVEC). Panels C and D: Representative section of a
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normal human adrenal gland displaying immunoractivity to an antibody against the type 2 fibroblast
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growth factor receptor (C) and a negative control section (D).
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Figure 6 This scheme summarizes the findings of this paper and that of Haase et al., (Haase et al., J Endocrinol 2007). In addition to other factors, endothelial cells synthesize and secrete basic fibroblast growth factor (bFGF) that binds to its receptor on adrenocortical cells and promotes β-catenin-dependent transcription. Corticotropin (ACTH) and angiotensin II (ATII) are
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physiologic regulators of adrenal steroidogenesis and bind to adrenal ACTH (MC2R) or ATII
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type 1 (AT1R) receptors, respectively.
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Endothelial cells regulate β-catenin activity in adrenocortical
Title:
cells via secretion of basic fibroblast growth factor Revised Version
β-catenin and bFGF
Authors:
Carolin Schwafertz , Sven Schinner , Markus C. Kühn , Matthias Haase
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Short Title:
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Amelie Asmus , Birgit Mülders-Opgenoorth , Ishrath Ansurudeen 4
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1,2
,
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, Peter J.
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Hornsby , Henning Morawietz , Elke Oetjen , Matthias Schott , Holger S. 1,6
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Willenberg
Highlights
endothelial cells induce β-catenin activity in adrenal cortical cells
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Wnt-signalling, cAMP, Akt or PKC pathways are not involved
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MEK, PI3-kinase and receptor tyrosine kinases are involved
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stimulation of β-catenin and StAR activity is partly mimicked by bFGF
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bFGF is produced and secreted by human umbilical cord endothelial cells
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•