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Endocrinology Vol. 145, No. 11 5177-5184
Copyright © 2004 by The Endocrine Society

The Epidermal Growth Factor-Like Growth Factor Amphiregulin Is Strongly Induced by the Adenosine 3',5'-Monophosphate Pathway in Various Cell Types

C. Christian Johansson, Arne Yndestad, Jorrit M. Enserink, Anne H. Ree, Pål Aukrust and Kjetil Taskén

Biotechnology Center of Oslo, University of Oslo (C.C.J., J.M.E., K.T.), Research Institute for Internal Medicine (A.Y., P.A.), and Section of Clinical Immunology and Infectious Diseases (P.A.), National Hospital, University of Oslo; and Department of Tumor Biology, Norwegian Radium Hospital (A.H.R.), N-0317 Oslo, Norway

Address all correspondence and requests for reprints to: Dr. Kjetil Taskén, Biotechnology Center of Oslo, University of Oslo, P.O. Box 1125, Blindern, N-0317 Oslo, Norway. E-mail: kjetil.tasken{at}biotek.uio.no.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We examined the cAMP-mediated regulation of the epidermal growth factor-like growth factor amphiregulin (AR) in T cells and observed a strong cAMP-induced up-regulation of AR mRNA in a time- and concentration-dependent manner independent of T cell activation. This regulation may be mediated in part through activation of a cAMP-responsive element in the AR promoter, because the cAMP-responsive element conferred cAMP responsiveness to a luciferase reporter in Jurkat TAg cells. Similar effects of AR mRNA induction were seen in T cells treated with cAMP-elevating agents such as prostaglandin E2 and forskolin as well as with the phosphodiesterase inhibitors rolipram and isobutylmethylxanthine. Furthermore, the induction of AR mRNA by cAMP was strongly suppressed by a protein kinase A type I-selective inhibitor, whereas treatment with an exchange protein directly activated by cAMP-specific agonist did not increase AR levels. In addition, an increase in AR gene transcripts by cAMP was seen in MCF-7 mammary carcinoma cells and H295R adrenal cells. Moreover, the potent cAMP-mediated induction of AR mRNA resulted in increased secretion (5-fold) of AR from T cells. Furthermore, supernatants from cAMP-stimulated T cells containing secreted AR induced phosphorylated MAPK in OVCAR-3 carcinoma cells. In conclusion, our data suggest that AR is under strong regulation by the cAMP pathway in various cell types, and that prostaglandin E2- and cAMP-induced AR secretion from T cells may be highly relevant in a microenvironment consisting of tumor cells and infiltrated immune cells, because AR by activating the MAPK pathway through a paracrine route may contribute to proliferation of tumor cells and thus add to neoplastic processes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AMPHIREGULIN (AR), SYNTHESIZED as a 252-amino acid transmembrane glycoprotein with two major soluble forms of 78 and 84 amino acids, is a heparin-binding growth factor originally isolated from conditioned medium of the human breast carcinoma cell line MCF-7 and belongs to the mammalian epidermal growth factor (EGF)-like family of growth factors that all bind and activate the EGF receptor (EGFR), ErbB1 (1, 2, 3, 4). Its name is derived from the dual role of this factor as both a positive and negative regulator of cell proliferation, which constitutes its major function. Indeed, AR promotes the growth of most cell types, including normal and neoplastic mammary epithelial cells, fibroblasts, and keratinocytes, whereas some other cell lines are growth inhibited (2, 5, 6, 7, 8, 9). Furthermore, AR has been shown in vitro to function in an autocrine manner to drive the proliferation of malignant cells of colon, breast, cervix, prostate, and pancreas (8, 10, 11, 12, 13). In addition, AR is commonly overexpressed in cancers of human colon, stomach, breast, and pancreas, in which the level of AR correlates with tumor progression and poor patient survival (11, 14, 15, 16, 17, 18, 19). Finally, it was previously reported that AR is a mitogen for adult mouse neural stem cells and may participate in the regulation of neural stem cell proliferation and neurogenesis (20). In contrast to all these reports, the literature is virtually void of data on the possible role of AR in the immune system.

cAMP-dependent protein kinase A (PKA) type I plays a key role in cell growth and differentiation. Enhanced levels of PKA type I are detected in tumor cells and overexpression of PKA type I has been correlated with poor prognosis in breast cancer patients (21). Furthermore, cAMP is a well established negative regulator of T cell receptor (TCR) signaling and inhibits T cell function through activation of PKA type I (reviewed in Ref. 22). Recently, we observed in a cDNA array screening of cytokine and cytokine-related genes that the mRNA expression of AR was highly up-regulated in anti-CD3-activated T cells treated with the cAMP agonist 8-(4-chloro-phenylthio)-cAMP (8-CPT-cAMP) (23). To elucidate a potential role of AR in T cells, we analyzed the expression of this growth factor in CD3+ T cells under basal and activated conditions as well as the ability of cAMP to modulate its expression. We show that the mRNA expression of AR is strongly up-regulated by cAMP in T cells in a time- and concentration-dependent manner, possibly mediated in part through activation of a cAMP-responsive element (CRE) in the AR promoter and independent of the T cell activation status. AR mRNA levels are increased by the cAMP-elevating agents prostaglandin E2 (PGE2) and forskolin as well as by the phosphodiesterase (PDE) inhibitors isobutylmethylxanthine (IBMX) and rolipram in both resting and activated T cells. Furthermore, cAMP-mediated up-regulation of AR gene transcripts was also observed in MCF-7 mammary carcinoma cells and H295R adrenal cells. In addition, we provide data indicating that the cAMP-induced up-regulation of AR is mediated by the PKA type I pathway and not by the Epac (exchange protein directly activated by cAMP) pathway. Importantly, we provide data indicating that T cell-secreted AR is able to activate the MAPK pathway in OVCAR-3 cells. Taken together, our data show that cAMP is a potent inducer of AR expression in and secretion from T cells as well as in cells of nonlymphoid origin. T cell-secreted AR may be highly relevant in a tumor microenvironment infiltrated with immune cells, considering the mitogenic effect of AR on many cell types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation of CD3+ T cells
Peripheral CD3+ T cells were purified as previously described (24). Briefly, peripheral blood mononuclear cells (PBMC), obtained from heparinized healthy human whole blood (Ullevaal University Hospital Blood Center, Oslo, Norway) by Isopaque-Ficoll (Lymphoprep, Nycomed Pharma AS, Oslo, Norway) gradient centrifugation, were mixed with beads coated with antibodies against CD14 (Dynabeads M-450 CD14, Dynal Biotech, Oslo, Norway) and CD19 (Dynabeads M-459 Pan B, Dynal Biotech) in a cell to bead ratio of 1:5 and incubated with rotation at 4 C for 1 h. The negatively selected cells consisted of more than 94% CD3+ T cells as assessed by flow cytometry.

T cell activation and stimulation
CD3+ T cells (5 x 106/ml) suspended in RPMI 1640 medium containing 2 mM L-glutamine (Invitrogen Life Technologies, Paisley, UK), 1% nonessential amino acids (Invitrogen Life Technologies), 1 mM sodium pyruvate (Invitrogen Life Technologies), and 100 U penicillin/streptomycin (Invitrogen Life Technologies) supplemented with 10% heat-inactivated (56 C for 45 min) and sterile-filtered (0.22 µm) fetal calf serum (Invitrogen Life Technologies), were incubated in a 5% CO2 incubator at 37 C in the presence of mouse IgG anti-CD3 antibodies (clone SpvT3b, 10 ng/ml; Zymed Laboratories, South San Francisco, CA). Subsequent cross-ligation of the TCR/CD3 complex was achieved by the addition of magnetic beads coated with sheep antimouse IgG (Dynal beads M-450 sheep antimouse IgG, Dynal Biotech) in a cell to bead ratio of 1:1. Cells were harvested after 3, 6, 12, and 24 h. When used, cAMP agonist (8-CPT-cAMP; 3, 10, 30, 100, 300, and 1000 µM or 8-bromo-adenosine-3',5'-cyclic monophosphoro-thioate, Sp isomer (Sp-8-Br-cAMPS); 300 µM; BioLog Life Science, Bremen, Germany), PKA type I-selective cAMP antagonist [8-bromo-cAMPS, Rp isomer (Rp-8-Br-cAMPS); 1000 µM; BioLog Life Science], PGE2 (25 µM; Sigma-Aldrich Corp., St. Louis, MO), forskolin (100 µM; Calbiochem, La Jolla, CA), IBMX (900 µM; Sigma-Aldrich Corp.), rolipram (10 µM; Sigma-Aldrich Corp.), isoproterenol (10 µM; Sigma-Aldrich Corp.), and Epac-specific agonist [8-CPT-2'-O-methyl-cAMP (8-CPT-2'-O-Me-cAMP); 30 µM; BioLogLife Sciences Institute] were added to cell cultures before anti-CD3 activation. Cells treated with vehicle (ethanol and dimethylsulfoxide) had no effect on AR mRNA induction as assessed by real-time quantitative RT-PCR.

AR immunoassay
CD3+ T cells (5 x 106/ml) were incubated in the absence or presence of 300 µM 8-CPT-cAMP. After 6, 12, and 24 h of culture, cell-free supernatants from these cultures were collected and stored at –80 C until additional analysis. Before immunoassays, samples were concentrated by using a Vivaspin concentrator (molecular weight cut-off at 5000) according to the manufacturer’s manual (Vivascience AG, Hanover, Germany). For determination of AR protein levels in the concentrated samples, a DuoSet ELISA Development kit for human AR (R&D Systems, Abington, UK) was used according to the manufacturer’s instructions.

Isolation of total RNA
Total RNA was isolated by using an RNeasy Mini Spin Kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual. Briefly, T cell lysates were homogenized and applied to an RNeasy mini spin column. After washes, total RNA was eluted by adding ribonuclease-free water onto the RNeasy silica gel membrane. RNase inhibitor (RNasin, Promega Corp., Madison, WI) was added to all RNA samples and stored at –80 C until additional analysis.

Real-time quantitative RT-PCR
Sequence-specific primers were designed for human AR (forward, 5'-ACTCGGCTCAGGCCATTATG-3'; reverse, 5'-CCAGAAAATGGTTCACGCTTC-3'; GenBank accession no. M30704) and human ß-actin (forward, 5'-AGGCACCAGGGCGTGAT-3'; reverse, 5'-TCGTCCCAGTTGGTGACGAT-3'; GenBank accession no. NM_001101) using the Primer Express software version 1.5 (Applied Biosystems, Foster City, CA). Quantification of mRNA was performed using the ABI PRISM 7700 (Applied Biosystems) as previously described (25). ß-Actin gene expression was analyzed using human ß-actin TaqMan predeveloped assay reagents (Applied Biosystems) and was used to adjust for unequal amounts of mRNA. All samples were analyzed in triplicate, standard curves were run on the same plate, and the relative standard curve method was used for calculation of relative gene expression.

Cell lines and cell culture
H295R adrenal cells and MCF-7 mammary carcinoma cells were cultured, 8-CPT-cAMP (100 µM)-stimulated, and RNA extracted as previously described (26, 27). OVCAR-3 (human ovarian carcinoma) cells and the human leukemic T cell line Jurkat TAg (a derivative of the Jurkat cell line stably transfected with the simian virus 40 large T antigen) were grown in RPMI 1640 medium as defined above for primary T cells.

Transfection and stimulation of cell cultures for AR promoter analyses
The firefly luciferase reporter constructs pGL3-C and pGL3-C{Delta}CRE containing the 5'-flanking region of the human AR gene were a gift from Dr. Sean B. Lee, who has shown that pGL3-derived AR constructs behave similarly to the pGL2 series described previously (28). AR is also expressed and modulated by cAMP in Jurkat TAg cells (as confirmed by real-time quantitative RT-PCR; not shown). Therefore, we used this T cell line for transfections and promoter analysis. For transfections, 20 x 106 Jurkat TAg cells were resuspended in 0.4 ml Opti-MEM (Invitrogen Life Technologies, Inc.) and transiently cotransfected with a total of 21 µg DNA (10 µg of one of the AR firefly luciferase plasmid constructs, 1 µg of the pRL-simian virus 40 plasmid (as an internal control) containing the Renilla luciferase gene, and empty vector made up to a total of 21 µg) by electroporation (250 V/cm, 950 µF) in cuvettes with a 0.4-cm electrode gap. Cells were then expanded in 20 ml complete RPMI medium in 75-cm2 culture flasks and incubated overnight. For stimulation, 8-CPT-cAMP (500 µM) was added the next day, and cells were cultured for another 10 h.

Luciferase assay
Jurkat TAg cells were collected by centrifugation, washed once in cold PBS, resuspended in reporter lysis buffer (Promega Corp.) and lysed for 30 min on ice. The cell lysates were centrifuged for 10 min at 13,000 rpm, and 5firefly and Renilla luciferase activities were measured in the supernatants using a dual luciferase reporter assay system (Promega) and a luminometer. Twenty microliters of lysate were used for both the firefly and Renilla luciferase readings. The firefly luciferase values were normalized to the Renilla luciferase values.

Western blotting
OVCAR-3 cells were plated, grown to 70% confluence, and stimulated for 5 min with EGF (50 ng/ml; Sigma-Aldrich Corp.), AR (50 ng/ml; R&D Systems), or cell-free supernatants from resting CD3+ T cells (5, 10, and 20 x 106/ml) that had been cultured for 24 h in the absence or presence of 8-CPT-cAMP (300 µM). In some experiments, OVCAR-3 cells were pretreated with PGE2 (100 µM) or forskolin (10 µM) for 5 min before EGF and AR stimulation. Subsequently, equal amounts of total OVCAR-3 cell lysates were loaded onto one-dimensional SDS-polyacrylamide 4–20% gradient gels (Bio-Rad Laboratories, Sundbyberg, Sweden), subjected to electrophoresis, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) by electroblotting. The membranes were blocked for 2 h in a solution containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20, and 5% BSA (for phospho-MAPK blot) or 5% milk [for protein kinase C{alpha} (PKC{alpha}) blot], and then incubated overnight at 4 C with a rabbit polyclonal phospho-specific antibody against phosphorylated human p44/42 MAPK (Cell Signaling Technology, Inc., Beverly, MA) or a mouse monoclonal antibody against human PKC{alpha} (Transduction Laboratories, Lexington, KY) in blocking solution. The membranes were washed in a solution containing 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.05% Tween 20. Immunoreactive proteins were visualized by Supersignal (Pierce Chemical Co., Rockford, IL) using horseradish peroxidase-conjugated rabbit or mouse secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) and subjected to autoradiography.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Time kinetics and cAMP-mediated regulation of AR gene expression in T cells
Recently, we observed in an array analysis that cAMP up-regulated AR mRNA in anti-CD3 activated CD3+ T cells (23). To examine whether T cell activation was a prerequisite for the observed cAMP-mediated regulation of AR, resting CD3+ T cells were treated with a cAMP agonist (8-CPT-cAMP; 300 µM). We found a strong (20-fold) induction of AR mRNA by cAMP (Fig. 1AGo), which indicates that cAMP-mediated induction of AR transcripts occurs independently of T cell activation. In contrast, T cell activation by cross-ligation of the TCR/CD3 complex with anti-CD3 did not induce AR levels (Fig. 1AGo). We also explored the effect of endogenous basal levels of cAMP on AR gene expression in resting and activated T cells. However, treatment of resting or anti-CD3-activated T cells with a cAMP antagonist (Rp-8-Br-cAMPS; 1000 µM) to block the effect of endogenous levels of cAMP did not significantly alter AR mRNA levels compared with untreated cells (Fig. 1AGo). We next examined the level of regulation of AR in activated T cells at various concentrations of 8-CPT-cAMP and found that AR is regulated in a concentration-dependent manner, with a maximal effect (50-fold) at 300 µM and a half-maximal effect at approximately 100 µM (Fig. 1BGo). Next, we examined temporal regulation of AR mRNA levels by cAMP treatment after triggering of the TCR/CD3 complex. CD3+ T cells from three healthy blood donors were resting or activated by cross-ligation of the TCR/CD3 complex for 3, 6, 12, and 24 h in the absence and presence of a cAMP agonist (8-CPT-cAMP; 300 µM), and AR gene expression was analyzed by real-time quantitative RT-PCR (Fig. 1CGo). T cell activation had little or no effect on levels of AR mRNA compared with resting T cells at the different time points. In contrast, cAMP treatment of activated T cells with 8-CPT-cAMP markedly increased AR gene expression by 15- to 50-fold, with maximal levels at 3 to 6 h. For additional gene expression studies, T cells were activated by anti-CD3 for 6 h.



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  		FIG. 1. Time- and cAMP-mediated regulation of AR gene expression in T cells. A, Peripheral CD3+ T cells were resting or anti-CD3 activated for 6 h in the absence and presence of 300 µM 8-CPT-cAMP (cAMP agonist) or 1000 µM Rp-8-Br-cAMPS (cAMP antagonist). Subsequently, total RNA was isolated from these cultures and subjected to real-time quantitative RT-PCR using gene-specific primers for AR and ß-actin. All AR PCR products (Ct-value) were normalized to the levels of ß-actin (Ct-value). B, Peripheral CD3+ T cells were anti-CD3 activated and treated with increasing concentrations of 8-CPT-cAMP (range, 0–1000 µM), cultured for 6 h, and then analyzed for AR gene expression as described in A. C, Peripheral CD3+ T cells were resting or activated by cross-ligation of the TCR/CD3 complex for 3, 6, 12, and 24 h in the absence and presence of 300 µM 8-CPT-cAMP. Next, total RNA was isolated and subjected to real-time quantitative RT-PCR for AR as described in A. The data presented are from two (A and B; mean ± half the range) or three (C; mean ± SEM) blood donors and represent the level of gene expression of AR relative to that of untreated resting cells.

 
Role of CRE in cAMP-mediated activation of AR promoter
It is well known that one of the ways in which cAMP may stimulate the expression of target genes is through a conserved CRE. A CRE site has been identified previously in the AR promoter (1). To unravel the role of the CRE site in the cAMP-mediated induction of AR transcription in T cells, Jurkat TAg cells were transfected with a pGL3-C construct containing the CRE site and the TATA box of the AR promoter (a schematic depiction of the firefly luciferase reporter plasmid is shown in Fig. 2AGo) or a pGL3-C{Delta}CRE plasmid, a construct that contains the AR promoter with the CRE site deleted. As shown in Fig. 2BGo, luciferase activity was increased (2.5-fold) by cAMP treatment (500 µM 8-CPT-cAMP) compared with untreated cells. Removal of the CRE site attenuated the cAMP-induced reporter activity. Notably, the pGL3-C construct showed a moderate reporter activity in the absence of 8-CPT-cAMP.



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  		FIG. 2. cAMP-mediated induction of the CRE in the AR promoter in Jurkat TAg cells. A, The pGL3-C firefly luciferase reporter plasmid, indicating the relative positions of the TATA box (–238 to –233) and the CRE site (–274 to –267) of the AR promoter are schematically depicted. The nucleotide numbers refer to the AR promoter sequence (1 ). B, Jurkat TAg cells (20 x 106/ml) were transfected with 10 µg AR promoter firefly luciferase reporter constructs of pGL3-C and pGL3-C{Delta}CRE (deletion of the CRE site) or empty vector alone together with 1 µg Renilla luciferase gene reporter plasmid and stimulated for 10 h in the absence and presence of 500 µM 8-CPT-cAMP. The firefly luciferase (Luc) values were determined in triplicate samples (mean ± SEM) and were normalized to the Renilla luciferase values. Luc/Renilla activity of the vector alone in the absence of cAMP was assigned a value of 1.

 
Regulation of AR mRNA levels via the PGE2-adenylate cyclase pathway
To examine whether AR is regulated via a G protein-coupled receptor-adenylate cyclase pathway we treated resting or activated CD3+ T cells with PGE2 (Fig. 3AGo) and forskolin (Fig. 3BGo), which are known to activate the adenylate cyclase. In addition, to inhibit the degradation of cAMP, T cells were treated with IBMX, a nonspecific PDE inhibitor, or rolipram, a PDE4 (cAMP-specific PDE particularly abundant in immune cells)-specific inhibitor. Figure 3AGo shows that both PGE2 (15-fold) and IBMX (60-fold) markedly up-regulated AR mRNA levels. When combined, PGE2 and IBMX together did not significantly induce the levels of AR any further, indicating that AR levels had reached a maximum with IBMX. As shown in Fig. 3BGo, both forskolin (15-fold) and rolipram (4-fold) up-regulated AR gene expression in resting and activated T cells compared with untreated T cells, suggesting that AR levels are regulated by changes in endogenous cAMP levels that are controlled in part by PDE4.



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  		FIG. 3. Regulation of AR gene expression by the PGE2-adenylate cyclase pathway. A, Resting peripheral CD3+ T cells were cultured in the absence and presence of 25 µM PGE2 or 900 µM IBMX alone or in combination for 6 h and then examined for levels of gene expression of AR by real-time quantitative RT-PCR as described in Fig. 1Go. B, Peripheral CD3+ T cells were either resting or anti-CD3 triggered in the absence and presence of 100 µM forskolin or 10 µM rolipram for 6 h, then examined for levels of gene expression of AR by real-time quantitative RT-PCR as described in Fig. 1Go. C, Peripheral CD3+ T cells were either resting or anti-CD3-triggered in the absence and presence of 300 µM Sp-8-Br-cAMPS (cAMP agonist) or 1000 µM Rp-8-Br-cAMPS (PKA type I- selective cAMP antagonist), alone or in combination, and 30 µM 8-CPT-2'-O-Me-cAMP (Epac-specific agonist) for 6 h, then examined for levels of gene expression of AR by real-time quantitative RT-PCR as described in Fig. 1Go. D, Peripheral CD3+ T cells were either resting or anti-CD3-triggered in the absence and presence of 10 µM isoproterenol (ß-adrenoceptor agonist) or 300 µM 8-CPT-cAMP for 6 h, then examined for levels of gene expression of AR by real-time quantitative RT-PCR as described in Fig. 1Go. The data presented are from two (A, B, and D; mean ± half the range) or three (C; mean ± SEM) blood donors and represent the level of gene expression of AR relative to that of untreated resting cells.

 
In many cell types, cAMP targets two independent signaling pathways: the PKA pathway and the Epac pathway. Epac is an exchange protein for the small GTPase Rap1, which has recently attracted much attention (29). To elucidate whether the cAMP-mediated regulation of AR gene expression is mediated by the PKA pathway or the Epac pathway, we made use of a PKA type I-selective inhibitor (Rp-8-Br-cAMPS) and an Epac-specific cAMP analog (8-CPT-2'-O-Me-cAMP) that activate Epac (30). Treatment of T cells with the cAMP agonist Sp-8-Br-cAMPS, which is nonselective for Epac and PKA, strongly induced AR mRNA levels in both resting and anti-CD3 activated T cells (30-fold), whereas in combination with the PKA inhibitor Rp-8-Br-cAMPS, this effect was substantially suppressed (Fig. 3CGo), suggesting a PKA type I-mediated regulation of AR. Indeed, treatment of T cells with the Epac-specific cAMP analog 8-CPT-2'-O-Me-cAMP did not induce AR mRNA levels (Fig. 3CGo), indicating that regulation of AR in T cells is not mediated through the Epac pathway. Notably, in these experiments treatment with anti-CD3 alone moderately induced AR mRNA levels (2-fold), whereas the addition of a cAMP antagonist (Rp-8-Br-cAMPS) to the anti-CD3-activated T cells suppressed AR levels to the basal levels observed in resting untreated cells. To elucidate whether AR levels are also induced via a ß-adrenoceptor pathway, resting or anti-CD3 activated T cells were treated with isoproterenol (an agonist for the ß-adrenoceptor). As shown in Fig. 3DGo, isoproterenol had only minor effects on AR mRNA levels compared with the AR induction by 8-CPT-cAMP (65-fold) or PGE2 (15-fold; Fig. 3AGo), indicating that the ß-adrenoceptor pathway is not a major route for regulating AR gene expression in T cells.

cAMP also regulates AR in nonlymphoid cell types
To explore whether the cAMP-mediated regulation of AR is restricted to T cells or occurs more generally, we screened various cell types for the expression of AR mRNA and regulation by cAMP by real-time quantitative RT-PCR. Figure 4Go shows that AR mRNA was present in both H295R adrenal cells and MCF-7 mammary carcinoma cells and that 8-CPT-cAMP (100 µM) induced the levels of AR 10- and 3-fold, respectively. In addition, time-dependent regulation of AR was examined in H295R cells, and maximal mRNA levels were seen after 3 h of 8-CPT-cAMP stimulation, indicating that the level of cAMP-mediated AR induction in MCF-7 cells might also be higher at shorter time points (Fig. 4Go). We also detected low, but cAMP-regulated, levels of AR mRNA in LN-CAP prostate carcinoma cells (not shown).



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  		FIG. 4. Regulation of AR mRNA levels by cAMP in MCF-7 mammary carcinoma cells and H295R adrenal cells. Total RNA from MCF-7 and H295R cells, which had been grown in the absence and presence of 100 µM 8-CPT-cAMP for 3, 5, and 13 h (H295R) and 12 h (MCF-7),was examined for levels of gene expression of AR by real-time quantitative RT-PCR as described in Fig. 1Go. Data are shown relative to untreated cells and were determined in triplicate samples (mean ± SEM).

 
cAMP increases the secretion of AR from T cells
Our finding that cAMP induces T cells to up-regulate AR mRNA levels inspired us to investigate whether treatment of T cells with cAMP also results in increased AR protein expression and secretion. Resting CD3+ T cells were cultured for 6, 12, and 24 h in the absence and presence of 8-CPT-cAMP (300 µM), and cell-free supernatants from these cultures were then assessed for AR protein levels at the indicated time points by ELISA. We observed an increased and sustained secretion (4- to 5-fold) of AR from T cells treated with cAMP (Fig. 5Go), reflecting the strong cAMP-mediated induction of AR gene transcripts.



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  		FIG. 5. cAMP-mediated regulation of T cell-secreted AR. A, Resting peripheral CD3+ T cells from three healthy blood donors were incubated for 6, 12, and 24 h in the absence and presence of 300 µM 8-CPT-cAMP. Next, cell-free supernatants from these cultures were assessed for levels of AR by ELISA. The fold increase in immunoreactive AR protein levels relative to that of untreated T cells is presented (mean ± SEM). B, Representative data from two blood donors from experiments described in A showing absolute levels (picograms per milliliter) of cAMP-mediated up-regulation of AR protein secretion (mean ± half the range).

 
Supernatant from cAMP-stimulated T cells induces MAPK phosphorylation in OVCAR-3 cells
Both p44 and p42 MAPK (Erk1 and Erk2) function in a protein kinase cascade that plays a critical role in the regulation of cell growth and differentiation (31, 32, 33, 34). MAPKs are activated by a wide variety of extracellular signals, including growth and neurotrophic factors, cytokines, hormones, and neurotransmitters. We wanted to elucidate whether T cell-secreted AR could confer a mitogenic stimulus to OVCAR-3 cells by inducing phosphorylated MAPK (P-MAPK). The OVCAR-3 ovarian carcinoma cell line expresses the EGFR and potently activates the MAPK pathway upon treatment with EGF. In an initial experiment we explored whether PGE2 and forskolin were able to induce P-MAPK in these cancer cells. OVCAR-3 cells were pretreated for 5 min with PGE2 or forskolin, then incubated for another 5 min in the absence and presence of EGF or AR, and subsequently examined for P-MAPK induction by Western blot analysis using a phospho-specific p44/42 MAPK antibody. PGE2 and forskolin did not induce P-MAPK to any extent compared with the P-MAPK levels induced by EGF and AR (Fig. 6AGo). Combination of EGF with PGE2, forskolin, or AR did not induce P-MAPK levels compared with the P-MAPK levels induced by EGF alone. Similar results were observed when AR was combined with PGE2 or forskolin (not shown). Next, CD3+ T cells were cultured for 24 h at increasing cell concentrations in the absence and presence of 8-CPT-cAMP (300 µM) to induce AR secretion. Subsequently, cell-free supernatants were harvested, and OVCAR-3 cells were stimulated with these supernatants for 5 min and assessed for phosphorylated MAPK induction as described above. As shown in Fig. 6BGo, the cell-free conditioned medium from T cell cultures stimulated with cAMP for 24 h induced a 2- to 5-fold induction of P-MAPK compared with controls. Supernatants from cAMP-stimulated T cell cultures grown at a cell density of 20 x 106/ml induced P-MAPK to the same extent as did EGF or AR. We also observed an induction of P-MAPK in human epidermoid carcinoma A431 cells stimulated with cell-free supernatants from T cells (not shown).



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  		FIG. 6. Supernatant from cAMP-stimulated T cells induces phosphorylated MAPK in OVCAR-3 cells. A, OVCAR-3 cells were pretreated with PGE2 (100 µM) or forskolin (Frsk; 10 µM) for 5 min, then incubated for another 5 min in the absence and presence of EGF (50 ng/ml) or AR (50 ng/ml). Next, equal amounts of total OVCAR-3 cell lysates were examined by immunoblotting using antibodies against P-MAPK (phospho-p44/42-specific antibody) and PKC{alpha}. B, Resting peripheral CD3+ T cells (5, 10, and 20 x 106/ml) were incubated for 24 h in the absence (–) and presence (+) of 300 µM 8-CPT-cAMP. After 24 h, cell-free supernatants from these T cell cultures were harvested and used for stimulation of OVCAR-3 cells for 5 min at 37 C. As controls, OVCAR-3 cells were also stimulated with recombinant EGF (50 ng/ml) and AR (50 ng/ml) for the same period of time. Next, total OVCAR-3 cell lysates were examined by immunoblotting as described in A.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are several lines of evidence that AR is overexpressed in cancers in which the level of AR correlates with tumor progression and poor patient survival (11, 14, 15, 16, 17, 18, 19). In this study we show that the EGF-like growth factor AR is expressed in and secreted from T cells and is strongly induced by the cAMP/PKA type I pathway at both the mRNA and protein levels, but not by the Epac pathway. Epac does not appear to be present in T cells (Enserink, J. M., unpublished observations), in agreement with the lack of effect of the Epac-specific cAMP analog on AR gene expression. The cAMP-mediated regulation possibly works in part through a CRE site located at –274 to –267 in the AR promoter (1). Recently, PGE2-mediated induction of AR expression in colon cancer cells through activation of the CRE site in the AR promoter was demonstrated (35). In this study we show a cAMP-mediated activation of the AR promoter through the CRE site in Jurkat TAg cells. The fact that we only observed a 2.5-fold increase in reporter activity by cAMP might be due to differences between Jurkat TAg cells and primary T cells. AR is expressed at much higher basal levels in Jurkat TAg cells (not shown) compared with primary T cells. Therefore, AR may be induced to a lesser extent by cAMP in these cells than in primary T cells. In addition, it has been reported that cAMP has differential effects on primary T cells vs. Jurkat cells, indicating that, for unknown reasons, Jurkat cells sometimes have a different response to cAMP than primary T cells (36). Furthermore, there may be other factors involved in the induction of AR expression in T cells. A recent report demonstrates in LS-174 cells a synergistic action between PGE2 and TGF-{alpha} or K-ras oncogene in the induction of AR expression (35). AR is regulated by PGE2, an inflammatory mediator that signals through prostaglandin EP2 and EP4 receptors expressed on T cells to yield cAMP, whereas the ß-adrenoceptor pathway does not seem to contribute to the induction of AR levels in T cells. In support of the cAMP-mediated regulation of AR in T cells, we observed a similar pattern of cAMP-mediated induction of AR mRNA in epithelially derived cells, such as MCF-7 mammary cells and LN-CAP prostate carcinoma cells, as well as in H295R adrenal cells. Recently, it was reported that LH-regulated ovulation is mediated via up-regulation of AR and other EGF-like factors (37). Our observation of cAMP-mediated regulation of AR in various cell types is in line with these data, because LH signals via several pathways, including that of cAMP.

The function of AR in T cells is not known, but AR could have both autocrine and paracrine effects when secreted from T cells. There was no effect on anti-CD3-induced T cell proliferation in the presence of recombinant AR (not shown), and EGFR expression was not detected on either purified T cells or PBMC (not shown), indicating that other cell types may be targets for the AR secreted from T cells. AR induced by cAMP-elevating agents and secreted from T cells present in a tumor microenvironment may promote cell growth and add to the neoplastic process, because the effect of AR on the proliferation of many cell types is mainly mitogenic. We observed that supernatants containing T cell-secreted AR induced P-MAPK in OVCAR-3 cells, suggesting that AR may promote the growth of ovarian carcinomas in a tumor microenvironment of ovarian cancer cells and infiltrated T cells. Conditioned medium from T cells may contain a complex mixture of factors, including that of AR. In support of our observations, PGE2 and forskolin, both inducing cAMP in OVCAR-3 cells (not shown), did not induce MAPK phosphorylation in OVCAR-3 cells, whereas stimulation of these cells with recombinant AR and EGF did. AR secreted from T cells might, in turn, act on the EGFR on neighboring cells. Indeed, it has been reported that AR induces tyrosine phosphorylation of the EGFR in SK-OV-3 ovarian cells, supporting our observation in OVCAR-3 cells (38). Moreover, it has been reported that activated monocytes secrete AR resulting in an AR-mediated tyrosine phosphorylation of the EGFR in A431 cells (39), supporting our P-MAPK data for these cells. Furthermore, AR has been implicated as a ligand for other receptor families, such as receptors for IGF, which is also expressed on T cells (40). It has been demonstrated that AR inhibits apoptosis through an IGF-I-dependent survival pathway in nonsmall cell lung cancer cells (41).

In conclusion, our data indicate that AR is under strong cAMP-mediated regulation in T cells as well as in cells of nonlymphoid origin, and the AR secreted from T cells in response to a cAMP stimulus may have a paracrine effect and a potentially strong impact on neoplastic processes. Through induction of the MAPK pathway, AR might promote tumor growth in a situation with tumor cells, such as ovarian carcinoma cells, in close context with immune cells, including T cells.


   Acknowledgments
 
RNA samples from H295R cells were contributed from experiments conducted in the laboratory by Dr. Helle K. Knutsen. RNA from LN-CAP prostate carcinoma cells was a generous gift from Dr. Kristin A. Taskén. AR promoter constructs were kindly provided by Dr. Sean B. Lee. The OVCAR-3 cell line was a kind gift from Dr. Johannes L. Bos. A431 cells were generously provided by Dr. Inger Helene Madshus. We are grateful for the skilful technical assistance of Gladys Tjørhom and Guri Opsahl.


   Footnotes
 
This work was supported by the Program for Advanced Studies in Medicine, the Research Council of Norway, University of Oslo (EMBIO Program), the Norwegian Cancer Society, the Novo Nordic Research Foundation Committee, the Anders Jahre’s Foundation, and European Union RTD Grant QLK3-CT-2002-02149.

Current address for J.M.E.: Ludwig Institute for Cancer Research, University of California, San Diego, California 92093-0669.

Abbreviations: AR, Amphiregulin; 8-CPT-cAMP, 8-(4-chloro-phenylthio)-cAMP; 8-CPT-2'-O-Me-cAMP, 8-CPT-2'-O-methyl-cAMP; CRE, cAMP-responsive element; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Epac, exchange protein directly activated by cAMP; IBMX, isobutylmethylxanthine; Luc, luciferase; PBMC, peripheral blood mononuclear cells; PDE, phosphodiesterase; PGE2, prostaglandin E2; PKA, cAMP-dependent protein kinase; PKC, protein kinase C; P-MAPK, phosphorylated MAPK; Rp-8-Br-cAMPS, 8-bromo-adenosine-3',5'-cyclic monophosphoro-thioate, Rp isomer; Sp-8-Br-cAMPS, 8-bromo-adenosine-3',5'-cyclic monophosphoro-thioate, Sp isomer; TCR, T cell receptor.

Received February 23, 2004.

Accepted for publication July 13, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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