Transactivation of EGFR by prostaglandin E₂ receptors: a nuclear story?

Abstract

The pharmacological modulation of hypoxia-inducible factor-1α (HIF-1α) and HIF-1α-regulated vascular endothelial growth factor-A (VEGF-A) in the kidney has therapeutic interest. Although it is assumed that prostaglandin E₂ (PGE₂) exerts its biological effects from the extracellular medium through activation of EP receptors located at the cell membrane, we have shown in human renal proximal tubular HK-2 cells (and other cell lines) that intracellular PGE₂ regulates the expression of HIF-1α expression and the production of VEGF-A. Here, we have found—through experiments involving EP receptors agonists, EP receptor gene silencing and inhibition of the prostaglandin uptake transporter—that these biological effects of PGE₂ are mediated by intracellular EP2 receptors. In sharp contrast with cell membrane EP2, whose activation results in increased production of cAMP, intracellular EP2 signaling was independent of cAMP. Instead, it involved c-src-dependent transactivation of epidermal growth factor receptor, which led to p38/ERK1/2-dependent activation of mitogen- and stress-activated kinase-1 (MSK-1) and to MSK-1-dependent-histone H3 phosphorylation and transcriptional up-regulation of retinoic acid receptor-β. Even more important, this signaling pathway was fully reproduced in nuclei isolated from HK-2 cell, which highlights the relevance of nuclear EP receptors in the up-regulation of HIF-1α. These results open the possibility that signal cascades that proceed entirely in the cell nucleus might be responsible for several PGE₂ effects that are assumed to be due to cell membrane EP receptors.

Electronic supplementary material

The online version of this article (doi:10.1007/s00018-014-1802-1) contains supplementary material, which is available to authorized users.

Introduction

Transcription factor hypoxia-inducible factor (HIF) supports tissue survival in hypoxia by regulating the expression of gene products that are involved in cellular energy metabolism, angiogenesis, erythropoiesis and other biological processes [1]. These products include enzymes involved in glucose uptake and metabolism, carbonic anhydrase IX, erythropoietin and vascular endothelial growth factor (VEGF) [2]. HIF expression is also increased in normoxia: growth and coagulation factors, hormones, stress factors and inflammatory mediators such as prostaglandin E₂ (PGE₂), cytokines, interleukin-1β, tumor necrosis factor-α, nitric oxide and reactive oxygen species increase the expression of HIF [3, 4]. These agents increase HIF transcription and/or translation [3, 5] but not HIF protein stability.

Activation of HIF-1α and production of VEGF-A in renal tissue and, more specifically, in renal epithelial cells may have beneficial [3, 5–10] or harmful [11–15] effects.

The final result is possibly dependent on the cell type, the pathological context and the stage of the disease.

We have previously found in human proximal tubular HK-2 cells, but also in other cell lines, that HIF-1α expression and VEGF-A production increase upon treatment with PGE₂ or PGE₂-increasing agents [16]. PGE₂ is synthesized by cyclo-oxygenases and prostaglandin E synthases from arachidonic acid, that is released from plasma membrane phospholipids by members of the phospholipase A₂. The biological actions of PGE₂ have been attributed to its interaction with plasma membrane EP receptors. They consist of four G-protein coupled receptors (GPCR) designated EP1, EP2, EP3 and EP4 [17]. Early studies indicated that EP1 and EP3 receptors are coupled to G_αq to activate Ca²⁺ signaling and G_i to inhibit adenylyl cyclase, respectively, whereas EP2 and EP4 receptors couple to G_αs to stimulate adenylyl cyclase [18, 19].

The current view on prostanoid signaling is as follows. Prostanoids, once formed, are quickly released to the outside of cells and therefore, they act as autocrine or paracrine mediators in the vicinity of their sites of production to maintain local homeostasis [20]. However, it has been identified a prostaglandin uptake transporter (PGT) that mediates the influx of prostaglandins into the cells [21–24]. According to the current view, PGT would only contribute to signal termination and metabolic clearance of prostaglandins through their selective uptake across the plasma membrane followed by non-selective oxidation within the cell [21]. However, recent studies [16, 25–31], have disclosed that intracellular PGE₂ has biological effects, so that PGT appears to provide at least one mechanism whereby PGE₂ and other prostaglandins can be delivered to an intracellular site of action. Accordingly, inhibition of PGT with bromocresol green or bromosulfophtalein results in prevention of PGE₂-mediated effects [16, 27, 30, 31]. The cell nucleus is particularly important in this context: recent studies have disclosed that the nuclear envelope plays a major role in signal transduction cascades: functional EP receptors can be localized at the nuclear membranes of a variety of cell types and tissues [16, 25–27]. The same is true for phospholipase A₂, cyclo-oxygenases and prostaglandin E synthase [32]. Thus the cell nucleus has to be considered as a possible intracellular location for PGE₂ production and for the initiation of signal transduction cascades dependent on EP receptors.

We have previously found in human proximal tubular HK-2 cells, but also in other cell lines [16] that PGE₂ increases HIF-1α expression and VEGF-A production through an intracrine mechanism. It involves the transport of PGE₂ to the inside of the cell and the transactivation of epidermal growth factor receptor (EGFR) by a subset of intracellular EP receptors. The term transactivation refers to this nonclassical mode of signaling system cross-talk (in distinction to receptor activation induced by cognate ligands, e.g. the activation of EGFR by EGF). According to our studies, EGFR transactivation results in ERK1/2-, p38 MAPK-dependent phosphorylation of MSK-1. This leads to phosphorylation of Ser¹⁰ in histone H3, activation of retinoic acid receptor β (RARβ) gene transcription and RARβ-dependent transcriptional up-regulation of HIF-1α. Finally, the production of VEGF-A increases as a result of HIF-1α activity in PGE₂-treated HK-2 cells [16, 30, 31]

In the present work we had three main objectives: (1) to identify the intracellular EP receptor(s) responsible for the increase in HIF-1α expression and VEGF-A production induced by PGE₂ in HK-2 cells, (2) to analyze the mechanism through which the identified EP receptor(s) transactivate EGFR and (3) to assess the role of nuclear EP receptors in the signaling cascade leading to intracellular PGE₂-induced RARβ up-regulation.

Materials and methods

Reagents

Selective agonists of EP receptors, ONO-DI-004 for EP1, ONO-AE1-259-01 for EP2, ONO-AE-248 for EP3 and ONO-AE1-329 for EP4 were a generous gift of Ono Pharmaceutical Co., Ltd. (Osaka, Japan). Prostaglandin E₂ (PGE₂), butaprost, PF04419948, AG1478, PD98059, SB203580 and H89 were purchased from Sigma (St. Louis, MO, USA). Nitrocellulose membrane was from Bio-Rad (Hercules, CA, USA). Enhanced chemiluminescence ECL detection system was from Amersham Biosciences (Airlington, Heights, IL, USA). Antibody against HIF-1α was purchased from BD Biosciences (Palo Alto, CA, USA), rabbit anti-phospho-EGFR, rabbit anti phospho-p38, rabbit antiphospho-erk1/2, rabbit anti-EGFR, mouse anti-src, rabbit anti-nucleoporin p62 and rabbit anti-phospho-MSK-1 antibodies were from Santa Cruz Biotechnology (Temecula, CA, USA). Rabbit anti-RARβ was from Abcam (Cambridge, UK), rabbit anti-H3,rabbit anti-phospho-src and anti-phospho-Histone 3 (Ser¹⁰) were from Cell Signaling (Danvers, MA, USA), rabbit anti-EP2 was from Cayman and anti-β-actin antibody was from Sigma.

Cell culture and treatments

Human proximal tubular epithelial (HK-2), was purchased from American Type Culture Collection (Rockville, MD, USA). HK-2 cells were maintained in DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), 1 % penicillin/streptomycin/amphotericin B and 1 % glutamine (Invitrogen, Carlsbad, CA, USA) and 1 % Insulin–Transferrin–Selenium (Sigma, St. Louis, MO, USA). The culture was performed in a humidified 5 % CO₂ environment at 37 °C. In all experiments, cells were plated at 70–90 % confluence and 24 h later they were treated with 1 µM PGE₂, 0.5 µM EP2 agonist or 1 µM EP(1/3/4)agonists for different periods of time, 10 µM AG1478, 1 µM H89, 1.6 µM PF04419948, 20 µM PD98059, 10 µM SB203580; 10 µM PP2, were added 1 h before the treatment. Samples were immediately analyzed except samples for the determination of the production of VEGF-A, which were stored at −80 °C until analyzed.

Protein isolation and western blotting

HK-2 cells were stimulated for different time periods, washed twice with ice-cold PBS and then harvested, scraped into ice-cold PBS, and then pelleted by centrifugation at 500×g for 5 min at 4 °C. Cells were kept on ice for 30 min and homogenized in a solution containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 % Triton X-100, 0.5 % sodium deoxycholate and protease inhibitors. Proteins from cell lysates were denatured by heating. Then, they were resolved by 10 % SDS-PAGE, and blotted onto a nitrocellulose membrane (BioTrace/NT) overnight in 50 mM Tris–HCl, 380 mM glycine, 0.1 % SDS, and 20 % methanol. Mouse anti-HIF-1α (1:1,000), rabbit anti-RARβ (1:1,000), rabbit anti-phospho EGFR (1:500) rabbit anti-phospho-c-src (1:1,000), rabbit anti-phospho-erk1/2 (1:1,000), anti-phospho-MSK-1 (1:1,000), rabbit phospho H3 (Ser¹⁰) (1:5,000), rabbit anti-EP2 (1:5,000), rabbit anti-EGFR (1:3,000), mouse anti-c-src (1:250) and mouse anti-nucleoporin p62 (1:10,000) antibodies were added followed by incubation for overnight at 4 °C. After treatment for 1 h at room temperature with the corresponding secondary antiserum (1:4,000), the signals were detected with enhanced chemiluminescence reagent using β-actin antibody (1:25,000) as loading control.

Cell transfection

Transient transfection with reporter luciferase plasmid tk-β-RARE-Luc (the plasmid, which contains a part of the RARE sequence from the promoter of the human RARβ gene, was a generous gift of Prof. Ronald Evans, The Salk institute, CA, USA) was performed as follows: 2.5 × 10⁶ cells per well were plated in 6-well plates 24 h before transfection. Cells were incubated 12 h at 37 °C with 1 ml OptiMEM (Invitrogen, CA, USA) containing complexes of 5 μl lipofectamine (Invitrogen, CA, USA), 1 μg human tk-β-RARE-Luc and 1 μg renilla luciferase reporter as an internal control. Transfected cells were next incubated with complete growth medium for 24 h. Finally, firefly luciferase activity of the tk-β-RARE-Luc reporter was measured with a Lumat LB9506 luminometer (Berthold Technologies, Herts, UK) and normalized against renilla luciferase activity using the dual-luciferase reporter assay system (Promega, Madison, WI, USA).

Transient transfection with pCRE-SEAP (BD Biosciences) was performed as above to measure the binding of transcription factors to CRE, providing a direct measurement of activation for this pathway. The secreted SEAP enzyme was assayed directly from lysated cell using the Sensolyte pNPP Alkaline phosphatase reporter gene assay (AnaSpec; Fremont, CA, USA) according to the manufacturer’s instructions.

For EP2 inhibition, we used EP2 siRNA (Santa Cruz, CA, USA) and scramble siRNA (Applied Biosystem) as a control. HK-2 cells at 70 % of confluence were transfected with 20 nM EP2 siRNA or 20 nM scramble siRNA. According to the manufacturer’s protocol, we used Lipofectamine 2000 (Invitrogen) to get the transfection. Transfected cells were submitted after 48 h and EP2 expression was evaluated by immunofluorescence.

Isolation of histone proteins

For the isolation of histone proteins, HK-2 cells were seeded into p100 plates (1.5 × 10⁶ cells per well). After the deprivation, cells were homogenized in 0.3 ml nuclear preparation buffer (10 mM Tris–HCl (pH 7.6). 150 mM NaCl, 1.5 mM MgCl₂, 0.65 % NP-40, 1 mM PMSF and protease inhibitors cocktail). Nuclei were recovered by centrifugation at 1.5×g for 15 min at 4 °C. Pellets were resuspended in 0.2 ml resuspended buffer: 10 mM Tris–HCl (pH 7.6), 3 mM MgCl₂, 10 mM NaCl, 1 mM PMFS and inhibitor proteases cocktail. Nuclei were extracted with 0.4 N H₂SO₄ to isolate total proteins. The samples were precipitated with trichloroacetic acid and then, incubated on ice for 1 h, and centrifuged at 12,000×g, for 10 min. Pellets were washed twice with ice-cold acetone and then resuspended in 20 mM Tris–HCl (pH 7.6).

cAMP measurement

HK-2 cells were pretreated with bromocresol green for 1 h, treated with PGE₂ or EP2 agonist for 30 min, and then, the cAMP levels were measured with a Cyclic AMP EIA Kit (Cayman) according to the manufacturer’s instructions. All assays were performed in triplicate and repeated three times.

Nuclear isolation

Cell nuclei were isolated as previously described [27]. Briefly: HK-2 cells were washed three times with ice-cold PBS, gently scraped, and pelleted at 500×g for 10 min. The cell pellet was resuspended in 300 µl lysis buffer (10 mM Tris–HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl₂, 100 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride), homogenized (100 gentle strokes) with a potter tissue grinders and then, centrifuged at 600×g for 10 min at 4 °C. The pellet was resuspended in 300 µl lysis buffer with 0.1 % (v/v) NP-40, left on ice for 10 min and pelleted thereafter at 600×g for 10 min. Nuclear pellet was resuspended in 20 mM HEPES/Tris (pH 7.0) containing 10 nM CaCl₂ and 300 mM sucrose for western blot and immunofluorescence assays.

Gene transcription assays and detection of RARβ RNA

Isolated nuclei were pretreated with different drugs and then, treated with PGE₂ or EP2 agonist for different time periods. Nuclei were incubated at 37 °C in a solution containing 10 mM Tris (pH 7.4), 135 mM KCl, 100 nM CaCl₂, 500 µM dNTPs, 100 U RNase inhibitor. RNA from isolated nucleus was isolated with Tri-Reagent (Sigma, St. Louis, MO, USA) according to the instructions of the manufacturer. One microgram of total RNA was reverse transcribed using 6 µg of hexamer random primer and 200 U M-MLV RT (Life Technologies) in the buffer supplied with the enzyme and supplemented with 1.6 µg/ml oligodT, 10 nM dithiothreitol (DTT), 40 U RNasin, and 0.5 mM of deoxyribonucleotides (dNTPs). Two microliters of RT reaction were then PCR amplified with specific primers for RARβ: sense 5′-GGGTAGGGTTCACCGAAAGT-3′; (antisense) 5′-CATGGGGGAATTCTGGTCCC-3′. PCR conditions were: denaturation at 94 °C for 5 min, followed by 36 cycles of 95 °C 1 min, 57 °C 1 min, 72 °C 1 min, and then a final cycle of 10 min at 72 °C. The signals were normalized with the β-actin gene expression level. The primers for β-actin were: sense 5′-AGA AGG ATT CCT ATG TGG GCG-3′ and antisense 5′-CAT GTC GTC CCA GTT GGT GAC-3′. The PCR products were separated by electrophoresis and visualized in 2 % agarose gels.

Immunofluorescence assay

Cells or isolated nuclei (on poly-l-lysine coated coverslips) were fixed with 2 % paraformaldehyde in PBS for 10 min at RT, permeabilized with 0.1 % Triton X-100 for 10 min, at RT and rinsed in PBS. Cells or nuclei were then incubated for 30 min with 4 % BSA in PBS to block nonspecific binding. Afterwards, cells or nuclei were incubated overnight at 4 °C with anti phospho-c-src (1:100), anti EPs (1:500) or anti phospho EGFR (1:50) and then, washed with PBS. Finally, cells were incubated with α-rabbit-Alexa-Fluor^® 488 (1:1,000) for 1 h in the darkness. Slides were then washed and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen). Detection was performed by confocal laser scan microscopy LEICA TCS-SL (Heidelberg, Germany).

Determination of VEGF secretion

HK-2 cells were placed in 24 well plates (5 × 10⁴ cells/well) for 24 h. After an incubation period of 48 h with the treatment, the medium was removed and kept at −80 °C for ELISA assays. VEGF was analyzed using human VEGF DuoSet (R&D Systems, Minneapolis, MN, USA).

Statistical analysis

Each experiment was repeated at least three times. The results are expressed as the mean ± SD. They were subjected to one-way analysis of variance (ANOVA) following by the Bonferroni’s test for multiple comparisons. The level of significance was set at P < 0.05. All data analyses were performed with GraphPad Prism 5.

Results

EP2 receptor is responsible for the PGE₂-induced sequence of events leading to HIF-1α-dependent production of VEGF-A

We investigated whether the sequence of events leading to PGE₂-induced increase in VEGF-A production in HK-2 cells could be assigned to a specific EP receptor subtype. To this end, we first studied the effect of selective agonists of EP receptors, ONO-DI-004 for EP1, ONO-AE1-259-01 for EP2, ONO-AE-248 for EP3 and ONO-AE1-329 for EP4, on EGFR tyrosine phosphorylation (the hallmark of EGFR activation). Western blot analysis showed that P-EGFR in HK-2 cells was only increased upon treatment with EP2 receptor agonist (Fig. 1a, upper panel). To confirm the involvement of EP2 receptor in PGE₂-induced EGFR tyrosine phosphorylation, expression of EP2 receptor was knocked down by transfection with siRNA EP2 (Fig. 1a, lower panel, inset) prior to incubating HK-2 cells with PGE₂. In these conditions, PGE₂-induced increase in EGFR tyrosine phosphorylation was abolished (Fig. 1a, lower panel).

Fig. 1.

EP2 receptor is responsible for the PGE₂-induced sequence of events leading to HIF-1α-dependent production of VEGF-A. a Upper panel Treatment with EP2 receptor agonist results in increased EGFR tyrosine phosphorylation. HK-2 cells were incubated for 5 min with selective agonists of EP receptors (1 μM ONO-DI-004 for EP1, 0.5 μM ONO-AE1-259-01 for EP2, 1 μM ONO-AE-248 for EP3 and 1 μM ONO-AE1-329) and tyrosine phosphorylation of EGFR was assessed by western blot analysis. Lower panel siRNA EP2 prevents PGE₂-induced increase in EGFR tyrosine phosphorylation. HK-2 cells were transfected with EP2 receptor siRNA or control siRNA (scramble). Thereafter, cells were incubated for 5 min with PGE₂ and phosphorylation of EGFR was assessed by immunofluorescence (the inset shows the diminished EP2 receptor expression upon treatment with its siRNA) or western blot analysis. b Upper panel, left Treatment with EP2 agonist increases the luciferase activity in cells transfected with a tk-β-RARE-Luc-driven reporter construct. HK-2 cells were transiently transfected with the plasmid. Then, they were incubated as above with selective EP agonists for 4 h and luciferase activity in cell lysates was measured. Center panel, left Up-regulation of RARβ and HIF-1α upon treatment with EP2 agonists. Cells were incubated for 8 h with 0.5 μM EP2 agonist ONO-AE1-259-01 or with 10 μM alternative EP2 agonist butaprost and expression of RARβ and HIF-1α was assessed by western blot analysis. Right PGE₂-induced up-regulation of RARβ and HIF-1α is prevented by siRNA EP2 receptor. HK-2 cells were transfected with EP2 receptor siRNA or control siRNA (scramble). Thereafter, cells were incubated for 8 h with PGE₂ and expression of RARβ and HIF-1α was assessed by western blot analysis. c Treatment with EP2 receptor agonist results in increased production of VEGF-A. Cells were incubated for 48 has in a and production of VEGF-A was quantified by ELISA. General information: (1) PGE₂ was 1 μM, (2) western blot experiments: equal protein loading was confirmed by probing with an anti-β-actin antibody. Each experiment was repeated three times and a representative photograph of the results is shown, (3) reporter gene experiments: luciferase activity was normalized by Renilla luciferase activity and expressed as relative luminescence units (RLU), (4) DAPI was used to stain cell nuclei in the immunofluorescence microscopy, (5) bars and error bars in graphs: each bar represents the mean ± SD of three different experiments. *P < 0.05 vs control

PGE₂-induced increase in P-EGFR results in a transcriptional increase in RARβ expression so that PGE₂ promotes an enhancement in the luciferase activity in HK-2 cells transfected with a reporter plasmid tk-β-RARE-Luc construct (from the retinoic acid response element in the RARβ gene promoter) [30]. To identify the EP receptor responsible for this effect, cells transfected with the reporter plasmid were treated with the specific ONO EP agonists. Again, only the EP2 receptor agonist reproduced the increasing effect of PGE₂ on the luciferase activity of cells transfected with the tk-β-RARE-Luc construct (Fig. 1b, left). This suggests that EP2 receptor is responsible for the transcriptional up-regulation of RARβ and the RARβ-dependent up-regulation of HIF-1α triggered by PGE₂ [30]. To confirm this prediction, expression of RARβ and HIF-1α was assessed by Western blot analysis in cells which were treated with two different selective EP2 receptor agonists (butaprost or ONO-AE1-259-01) or in PGE₂-treated HK-2 cells which were previously transfected with siRNA EP2. The results shown in Fig. 1b (center and right) supported the proposed role of EP2 receptor, given that expression of RARβ and HIF-1α was increased by the EP2 receptor agonist whereas the knockdown of the EP2 receptor blunted the up-regulation of RARβ and HIF-1α upon treatment with PGE₂.

Finally, experiments in which we measured the production of VEGF-A in response to specific ONO agonists for each EP receptor, indicated that, again, the EP2 receptor agonist was the only able to promote a rise in the production of VEGF-A in HK-2 (Fig. 1c).

Taken together, the results shown in Fig. 1, indicate that EP2 is the EP receptor responsible for the PGE₂-induced increase in VEGF-A production.

Activation of c-src by EP2 receptor mediates the transactivation of EGFR upon treatment with PGE₂ and is critical for the increase in the HIF-1α-dependent production of VEGF-A induced by PGE₂

Previous studies have shown that EGFR may be transactivated by EP2 receptor via Src. To assess the relevance of src in the mechanism through which EP2 increases the expression of HIF-1α and the production of VEGF-A in HK-2 cells, we studied the effect of EP2 agonist ONO-AE1-259-01 and PGE₂ on the phosphorylation of c-src and the effect of c-src inhibitor PP2 on EP2- and PGE₂-induced EGFR phosphorylation as well as on PGE₂-induced increase in the expression of RARβ and HIF-1α and the production of VEGF-A. Our results indicated (Fig. 2) (1) that c-src phosphorylation increased upon treatment with PGE₂ or EP2 agonist, (2) that PGE₂-induced c-src phosphorylation was prevented by transfection with siRNA EP2 receptor and (3) that PP2 prevented PGE₂-induced EGFR phosphorylation as well as the increase induced by PGE₂ in both the expression of RARβ and HIF-1α and the production of VEGF-A. These results indicate that activation of c-src by EP2 receptor mediates the effects of PGE₂ on the expression of HIF-1α and the production of VEGF-A in HK-2 cells.

Fig. 2.

Activation of c-src by EP2 receptor mediates the transactivation of EGFR upon treatment with PGE₂ and is critical for the increase in the HIF-1α-dependent production of VEGF-A induced by PGE₂. Upper panel Increase in c-src phosphorylation upon treatment with PGE₂ and its prevention by transfection with siRNA EP2 receptor. Left and center HK-2 cells were treated with PGE₂ and phosphorylation of c-src was studied by western blot analysis (left, inset treatment with EP2 agonist ONO-AE1-259-01) or immunofluorescence assay (center). Right HK-2 cells were transfected with siRNA EP2 receptor. Then they were treated for 1 min with PGE₂ and phosphorylation of c-src was studied by western blot analysis. Middle panel Inhibitor of c-src phosphorylation PP2 prevents PGE₂-induced increase in EGFR phosphorylation. HK-2 cells were incubated for 1 h with 10μM PP2 prior to being incubated for 5 min with PGE₂ or EP2 receptor agonist ONO-AE1-259-01. Lower panel Inhibitor of c-src phosphorylation PP2 prevents the up-regulation of RARβ and HIF-1α and the increase in VEGF-A production induced by PGE₂. HK-2 cells were treated with PP2 and PGE₂ as above and expression of RARβ and HIF-1α expression (left, 8 h incubation with PGE₂) and VEGF-A production (right, 48 h incubation with PGE₂) were quantified, respectively, by western blot or ELISA. General information: (1) PGE₂ was 1 μM and EP2 agonist ONO-AE1-259-01 was 0.5 μM, (2) western blot experiments: equal protein loading was confirmed by probing with an anti-β-actin antibody. Each experiment was repeated three times and a representative photograph of the results is shown, (3) DAPI was used to stain cell nuclei in the immunofluorescence microscopy, (4) bars and error bars in graphs: each bar represents the mean ± SD of three different experiments. *P < 0.05 vs. control

Intracellular EP2 receptors, in an AMPc-independent manner, are responsible for EGFR transactivation and downstream events leading to increased production of VEGF-Aupon treatment with EP2 receptor agonist

If ONO-AE1-259-01 were a substrate for the PGT transporter, BG should be able to prevent the activation by EP2 receptor agonist of the mechanism responsible for the increase in VEGF-A production. Our results (Fig. 3a) confirmed this prediction. The same was true when VEGF-A production was increased by EP2 receptor agonist butaprost, which suggested that the uptake of EP2 receptor agonists by the BG-sensitive PGT is required to trigger Src-dependent EGFR transactivation and the subsequent EGFR-dependent effects.

Fig. 3.

Intracellular EP2 receptors, in an AMPc-independent manner, are responsible for EGFR transactivation and downstream events leading to increased production of VEGF-Aupon treatment with EP2 receptor agonist. a Upper panel Increased phosphorylation of c-src, EGFR and histone H3 (Ser¹⁰) upon treatment with EP2 receptor agonist is prevented by bromocresol green (BG), an inhibitor of the PGT transporter. HK-2 cells were treated for 1 h with the inhibitor of prostaglandin uptake transport BG (50 μM) and then incubated for 5 min with EP2 receptor agonist ONO-AE1-259-01. Lower panel EP2 receptor agonist-induced up-regulation of RARβ and HIF-1α and production of VEGF-A are prevented by BG. HK-2 were treated as above and expression of RARβ and HIF-1α expression (left, 8 h incubation with ONO-AE1-259-01) and VEGF-A production (right, 48 h incubation with EP2 agonist ONO-AE1-259-01 or 10 μM alternative EP2 agonist butaprost) were quantified, respectively, by western blot analysis or ELISA. b Upper panel PGE₂/EP2 receptor-induced increase in cAMP intracellular levels and CRE activity are not prevented by BG. Left HK-2 cells were treated for 1 h with the inhibitor of prostaglandin uptake transport bromocresol green (50 μM) and then incubated for 30 min with PGE₂ or EP2 agonist ONO-AE1-259-01. HK-2 cells were lysed and cAMP was quantified by ELISA. Right Cells which were previously transfected with pCRE-SEAP were treated as above and the secreted SEAP enzyme was assayed directly from lysated cells as indicated in the “Materials and methods” section. Lower panel, left Pre-incubation with the inhibitor of c-AMP-dependent kinase H89 does not prevent EP2 agonist-induced EGFR phosphorylation. Cells were pre-incubated for 1 h with 10 μM H89 and then for 5 min with ONO-AE1-259-01. Right RARβ and HIF-1α are not up-regulated upon treatment of HK-2 cells with cAMP increasing agent forskolin. Cells were treated for 8 h with 0.1 μM forskolin. General information: (1) PGE₂ was 1 μM and EP2 agonist ONO-AE1-259-01 was 0.5 μM, (2) western blot experiments: equal protein loading was confirmed by probing with an anti-β-actin antibody. Each experiment was repeated three times and a representative photograph of the results is shown, (3) bars and error bars in graphs: each bar represents the mean ± SD of three different experiments. *P < 0.05 vs control

EP2 receptor is a stimulatory Gs GPCR. Therefore, its activation in the cell membrane by PGE₂ stimulates adenylate cyclase, resulting in elevation of cytoplasmic cAMP levels to initiate multiple downstream events [17]. To assess the relevance of this pathway in our results, we quantified by ELISA the production of cAMP and the activity CRE in HK-2 cells which were pre-incubated with BG before being treated with PGE₂ or EP2 receptor agonist. BG did not prevent the increase in cAMP and CRE activity induced by PGE₂ or EP2 agonist (Fig. 3b), which suggested that cAMP was not involved in EGFR transactivation by EP2 receptor and the subsequent EGFR-dependent effects found in PGE₂- or ONO-AE1-259-01-treated cells. This view was further supported by two facts: (1) pre-incubation with the inhibitor ofcAMP-dependent protein kinase H89 did not prevent the transactivation of EGFR induced by EP2 receptor agonist and (2) expression of RARβ and HIF-1α was unchanged in HK-2 cells treated with the cAMP increasing agent forskolin (Fig. 3b).

Overall, the results shown in Fig. 3 indicate that intracellular EP2 receptors, in acAMP-independent manner, are responsible for EGFR transactivation upon treatment with PGE₂ leading to increased production of VEGF-A.

Isolated cell nuclei reproduce the signaling cascade leading to the increase of HIF-1α expression and VEGF-A production in PGE₂-treated cells

As suggested in the Introduction, the cell nucleus might participate in the signal transduction cascades triggered by intracellular PGE₂. Therefore, we decided to investigate whether nuclei isolated from HK-2 expressed EP receptors, c-src, and EGFR. Figure 4a shows that this was the case indeed. Therefore, we hypothesized that EGFR transactivation by EP receptors may proceed entirely in the nucleus of PGE₂-treated HK-2 cells. In a series of experiments designed to test our hypothesis we found that treatment with either PGE₂ or EP2 receptor agonist resulted in increased phosphorylation of c-src and EGFR (Fig. 4b), and that PGE₂-induced EGFR phosphorylation was prevented by inhibitors of EP2 or c-src activation (Fig. 4b). These results agree with the view that nuclear EP2 receptors are able to transactivate EGFR in a c-src-dependent manner upon treatment with PGE₂. In HK-2 cells EGFR transactivation upon treatment with PGE₂ results in phosphorylation of ERK1/2 and p38 MAPK [31]. Therefore, we sought to determine in isolated nuclei treated with PGE₂ whether there was EGFR-dependent phosphorylation of ERK1/2 and p38 MAPK. Figure 4c (upper panel) confirmed our hypothesis: there was an AG1478-sensitive (and EP2 antagonist sensitive) increase in P-ERK1/2 and P-p38 MAPK upon treatment with PGE₂. These results strongly suggested that phosphorylation of EGFR in the cell nucleus gave rise to kinase-dependent signaling.

Fig. 4.

Isolated cell nuclei reproduce the signaling cascade leading to the increase of HIF-1α expression and VEGF-A production in PGE₂-treated cells. a Nuclei isolated from HK-2 cells express c-src, EGFR and EP receptors. Nuclei were isolated as described in “Materials and methods”. Expression of EP receptors was detected by immunofluorescence microscopy (inset) and expression of c-src, EGFR and EP2 receptors was investigated by western blot analysis. b Left and center Treatment of cell nuclei with PGE₂ or EP2 agonist results in increased phosphorylation of c-src and EGFR. Nuclei were treated with PGE₂ or EP2 agonist ONO-AE1-259-01. Right PGE₂- or EP2 agonist-induced increase in P-src and P-EGFR is prevented by EP2 antagonist or c-src inhibitor. Nuclei were pre-incubated for 1 h with 1.6 μM PF04419948 (antagonist of EP2 receptors) or 10 μM PP2 (c-src inhibitor), then they were treated for 5 min with PGE₂ or EP2 agonist ONO-AE1-259-01 and phosphorylation of c-src and EGFR was assessed by western blot analysis. c Upper panel Treatment of cell nuclei with PGE₂ increases P-ERK1/2 and P-p38 MAPK, which is prevented by inhibitor of EGFR activation AG1478. Nuclei were pre-incubated with 1 μM AG1478, then they were treated with PGE₂ and phosphorylation of ERK1/2 and p38 was assessed by western blot analysis. Lower panel Treatment of cell nuclei with PGE₂ results in increased phosphorylation of MSK-1 and histone H3. Nuclei were treated with PGE₂ and phosphorylation of MSK-1 and histone H3 (Ser¹⁰) was assessed by western blot analysis. d Treatment of cell nuclei with PGE₂ results in increased transcription of RARβ in an EP2-receptor antagonist-, MSK-1 inhibitor-sensitive manner. Upper panel Isolated nuclei, resuspended in a solution containing dNTPs (see “Materials and methods”), were treated up to 3 h with PGE₂or EP2 receptor agonist ONO-AE1-259-01 and expression of RARβ mRNA was assessed by semi-quantitative RT-PCR. Lower panel Isolated nuclei were resuspended as above and treated for 1 h with 1.6 μM EP2 receptor antagonist PF04419948 or 0.1 μM MSK-1 inhibitor H89. Then they were treated for 1 h with PGE₂ and expression of RARβ mRNA was assessed as above. General information: (1) PGE₂ was 1 μM and EP2 agonist ONO-AE1-259-01 was 0.5 μM, (2) western blot and RT-PCR experiments: equal protein or RNA loading was confirmed, respectively, by probing with an anti-β-actin antibody or by assessing the β-actin gene expression level. Each experiment was repeated three times and a representative photograph of the results is shown, (3) DAPI was used to stain cell nuclei in the immunofluorescence microscopy

If this is true, it follows that the remaining steps of the pathway involved in PGE₂-induced HIF-1α up-regulation in HK-2 cells (i.e. phosphorylation of MSK-1 leading to histone H3 phosphorylation and transcriptional up-regulation of RARβ [31]) may also well take place in the cell nucleus. To test this hypothesis, we first studied in isolated nuclei the effect of PGE₂ on the phosphorylation of MSK-1 and histone H3 (at Ser¹⁰). As expected, the treatment resulted in an increased phosphorylation of all the items studied (Fig. 4c, lower panel).

Owing to the relevant role of MSK-1-induced H3 phosphorylation at Ser¹⁰ in the activation of the RARβ gene transcription by PGE₂ [31], we finally studied whether PGE₂ was also able to up-regulate RARβ mRNA in isolated nuclei in an EP2 receptor-dependent manner. Our results indicated that both PGE₂ and EP2 receptor agonist ONO-AE1-259-01 determined an increase in RARβ mRNA expression in the cell nuclei (Fig. 4d, upper panel). In addition, pre-treatment with an EP2 receptor antagonist or with an inhibitor of MSK-1 prevented the PGE₂-induced increase in RARβ mRNA (Fig. 4d, lower panel).

The results shown in Fig. 4, indicate that isolated cell nuclei reproduce the signaling cascade leading to the increase of HIF-1α expression and VEGF-A production in PGE₂-treated cells, suggest that the cell nucleus is a possible location for the intracellular action of PGE₂.

Discussion

Among the renal cells, proximal tubule epithelial cells are the most populous cell type and they play a relevant role in the pathogenesis of a vast array of kidney diseases, acute and chronic [33]. We have found that PGE₂ increases HIF-1α expression and VEGF-A production through an intracrine mechanism involving the transactivation of EGFR by intracellular EP receptors [30]. Here we have demonstrated that these effects are due to intracellular EP2 receptors and that the cell nucleus reproduces the EP2-dependent signaling pathway. Therefore, our data raise the issue that a number of PGE₂ effects in the proximal tubule (and perhaps in other locations) that are being currently attributed to cell membrane EP receptors might be the consequence of signal cascades that proceed entirely in the cell nucleus.

Intracrine signaling refers to a process whereby a ligand, originating within a target cell or taken up from the extracellular milieu, acts upon intracellular receptors (including those on the nucleus). Although still poorly understood, the diverse functions exerted by agonists and hormones acting at intracellular GPCRs suggest that intracrine signaling may play important roles distinct from those of the same receptors activated at the cell surface [34]. This view is supported by our results: EP2-induced increase in HIF-1α/VEGF-A was prevented by inhibitor of prostaglandin uptake transporter BG, which strongly suggests that only intracellular EP2 receptors were involved in these EP2 effects. EP receptors and other GPCR have not been universally detected in the nucleus [27] and, at most, our results in isolated nuclei only would apply to those types of cells that do express nuclear EP2 receptors. As a generalization of our results, we speculate the following for cells expressing nuclear EP2 receptors: that the biological responses to PGE₂ mediated by EP2 do not arise solely at the cell surface but may result from the integration of extracellular and intracellular (nuclear) signaling.

We have previously shown that intracrine PGE₂ signaling through intracellular EP receptors is required for the increase in HIF-1α/VEGF-A induced by hypoxia and other stimuli [16, 30]. It is unclear if these stimuli can induce nuclear PGE₂ signaling cascade and further studies should be performed to clarify this relevant question.

Inhibitor of prostaglandin uptake transporter bromocresol green (that has allowed us to discriminate the participation of cell membrane and intracellular EP receptors), not only prevented the intracellular actions of PGE₂ but also the effects of EP2 agonists. This suggests that perhaps several other EP agonists and antagonists may also be transported to inside of the cell and activate intracellular EP receptors. If the researchers are unaware of this possibility, they will probably assign to cell membrane EPs the results of experiments with EP agonists and antagonists. Therefore, the actual extension to which intracellular EP receptors may have contributed to PGE₂ effects attributed to cell membrane EP receptors remains to be determined.

We provide here evidence on the role of intracellular EP2 receptors in c-src-mediated EGFR transactivation during intracrine PGE₂ signaling (Fig. 3a). In light of these results, the assumption that cell membrane EP2 receptors are responsible for c-src-mediated EGFR transactivation in other experimental settings [35–38] should be re-evaluated. A related issue is the role of cAMP in intracrine signaling through EP2 receptor: cell membrane EP2 is positively coupled through Gs to cAMP production [17] and, in fact, it has been previously shown that c-src-mediated EGFR transactivation by EP2 is cAMP-dependent [35–37]. On the contrary, we have not found any connection between cAMP signaling and EGFR transactivation by intracellular EP2 receptors: the increase in EGFR-P upon treatment with EP2 receptor was insensitive to cAMP-dependent kinase inhibitor H89. Furthermore BG did not inhibit EP2-induced cAMP-dependent signaling, which suggests that only cell membrane EP2 receptors, but not intracellular EP2 receptors (the actual effectors), are coupled to cAMP production (Fig. 3b). Worth to mentioning, EP2 ligand (butaprost)-induced c-src-dependent EGFR transactivation was independent of G protein/cAMP in mouse keratinocytes [38]. Stimulation of EP2 led to the formation of a β-arrestin–Src complex (instead of β-arrestin-mediated internalization of EP2) that contributed to the transactivation of EGFR. The role of intracellular β-arrestin–Src complexes in intracellular EP2-induced EGFR transactivation in HK-2 cells remains to be elucidated.

There are two subcellular locations, the nucleus and the mitochondrion, in which EGFR has been detected [39]. Therefore, they are potential sites in which activation of intracellular EP2 receptors may result in EGFR transactivation. Here we have chosen the cell nucleus because EGFR has been much more studied here than in its mitochondrial location. Our results indicated that (1) treatment with PGE₂ or EP2 receptor agonist resulted in increased phosphorylation of c-src and EGFR (Fig. 4b), and that PGE₂-induced EGFR phosphorylation was prevented by inhibitors of EP2 or c-src activation (Fig. 4b), (2) PGE₂ induced EP2-, EGFR-dependent phosphorylation of ERK1/2 and p38 MAPK (Fig. 4b), (3) PGE₂ increased phosphorylation of MSK-1 and histone H3 (at Ser¹⁰) and (4) PGE₂ up-regulated RARβ mRNA (Fig. 4d) in an EP2-, MSK-1-dependent manner. Therefore, the cell nucleus has the potential to reproduce the signaling involved in the increase in HIF-1α and VEGF-A in PGE₂-treated HK-2 cells.

The results shown in Fig. 4 have several interesting implications. Regarding EGFR, the current view is that internalization of plasma membrane-activated EGFR gives rise to nuclear EGFR. Consequently nuclear EGFR appears in the phosphorylated form and it retains its tyrosine kinase activity [39]. However, our results (Fig. 1a, b) suggest that not all nuclear EGFR is in the P-EGFR form since phosphorylation of nuclear EGFR in HK-2 cells or nuclei isolated from them increases upon treatment with PGE₂. A related issue is whether nuclear EGFR retains its tyrosine kinase activity: EGFR inhibitor prevented the increase in P-ERK1/2 and P-p38 upon treatment of isolated nuclei with PGE₂ (Fig. 4c). This strongly suggested that phosphorylation of EGFR in the cell nucleus gave rise to kinase-dependent signaling. Although this piece of evidence agrees with the view that nuclear EGFR retains its tyrosine kinase activity [40], further studies will be required to confirm it.

Different roles for plasma membrane and nuclear receptors for PGE₂ have been proposed on the basis of studies on EP3 receptors in brain microvascular endothelial cells [27]. In these cells, distinct signaling pathways and functions are activated by plasma membrane and nuclear EP3 receptors: plasma membrane EP3 elicits immediate physiological actions (vasomotor effects), whereas the nuclear EP3, that participates in the intracrine mode of action of PGE₂, conveys gene regulation (endothelial nitric oxide synthase expression). Other intracrine genomic effects of PGE₂ through its perinuclear receptors, are the increase in the transcription of mitogenic transcription factor c-fos (via EP1) [25] and inducible nitric oxide synthase (via EP3) [26]. The view that nuclear EP receptors but not plasma membrane EP convey gene regulation in the intracrine mode of action of PGE₂ is reinforced by our own findings on the regulation of RARβ expression by PGE₂: while nuclear EP2 receptors mediate the increase in the transcription of RARβ upon treatment with PGE₂ of isolated nuclei (Fig. 4d), cell membrane EP receptors do not participate in the regulation of RARβ expression by PGE₂ in HK-2 cells [30]. The intracrine effects of PGE₂ on the cell nucleus of HK-2 cells open new avenues in the physiology of PGE₂ signaling at the proximal tubule and provide new vistas in the pharmacological modulation of HIF-1α/VEGF-A with therapeutic purposes.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Figure S1. EP2 receptor is responsible for the PGE ₂ -induced sequence of events leading to HIF-1α-dependent production of VEGF-A: quantification of the experiments involving Western blot analysis a) Upper panel: Treatment with EP2 receptor agonist results in increased EGFR tyrosine phosphorylation. * P < 0.05 vs control and other EP agonists. Lower panel: siRNA EP2 prevents PGE ₂-induced increase in EGFR tyrosine phosphorylation. * P < 0.05 vs other groups ** P < 0.05 vs control. b) Left: Up-regulation of RARβ and HIF-1α upon treatment with EP2 agonists.* P < 0.05 vs control. Right: PGE ₂-induced up-regulation of RARβ and HIF-1α is prevented by siRNA EP2 receptor * P < 0.05 vs other groups. General information: Data are mean ± SD (fold change over control/scramble) of the densitometric analysis of three different experiments in which protein expression was normalized to β-actin.

Figure 2 Activation of c-src by EP2 receptor mediates the transactivation of EGFR upon treatment with PGE ₂ and is critical for the increase in the HIF-1α-dependent production of VEGF-A induced by PGE ₂ : quantification of the experiments involving Western blot analysis. Upper panel: Increase in c-src phosphorylation upon treatment with PGE ₂ and its prevention by transfection with siRNA EP2 receptor. Middle panel. Inhibitor of c-src phosphorylation PP2 prevents PGE ₂-induced increase in EGFR phosphorylation. Lower panel. Inhibitor of c-src phosphorylation PP2 prevents the up-regulation of RARβ and HIF-1α induced by PGE ₂. General information: Data are mean ± SD (fold change over control) of the densitometric analysis of three different experiments in which protein expression was normalized to β-actin. * *P< 0.05 vs control/scramble; **P < 0.05 vs other groups.

Figure 3 Intracellular EP2 receptors, in an AMPc-independent manner, are responsible for EGFR transactivation and downstream events leading to increased production of VEGF-A upon treatment with EP2 receptor agonist: quantification of the experiments involving Western blot analysis. a) Upper panel: Increased phosphorylation of c-src, EGFR and histone H3 (Ser ¹⁰ ) upon treatment with EP2 receptor agonist is prevented by bromocresol green (BG), an inhibitor of the PGT transporter Lower panel: EP2 receptor agonist-induced up-regulation of RARβ and HIF-1α is prevented by BG. b) Upper panel: Left: Pre-incubation with the inhibitor of c-AMP-dependent kinase H89 does not prevent EP2 agonist-induced EGFR phosphorylation. Right: RARβ and HIF-1α are not up-regulated upon treatment of HK-2 cells with cAMP increasing agent forskolin. Lower panel: The pharmacological agentsforskolin and H89 were effective in increasing or preventinf, respectively basal or PGE2-induced increase in CRE activity. General information: Data are mean ± SD (fold change over control) of the densitometric analysis of three different experiments in which protein expression was normalized to β-actin. *P < 0.05 vs other groups.

Figure 4: Isolated cell nuclei reproduce the signaling cascade leading to the increase of HIF-1α expression and VEGF-A production in PGE ₂ -treated cells: quantification of the experiments involving Western blot analysis or RT-PCR. a) Upper and middle panels Treatment of cell nuclei with PGE ₂ or EP2 agonist results in increased phosphorylation of c-src and EGFR, which is prevented by EP2 antagonist or c-src inhibitor.* P < 0.05 vs control; **P < 0.05 vs other groups. Lower panel: left: Treatment of cell nuclei with PGE ₂ increases P-ERK1/2 and P-p38 MAPK, which is prevented by inhibitor of EGFR activation AG1478. *P < 0.05 vs other groups. Right:Treatment of cell nuclei with PGE ₂ results in increased phosphorylation of MSK-1 and histone H3* P < 0.05 vs controlb) Treatment of cell nuclei with PGE ₂ results in increased transcription of RARβ in an EP2-receptor antagonist-, MSK-1 inhibitor-sensitive manner. * P < 0.05 vs control; **P < 0.05 vs other groups. General information: Data are mean ± SD (fold change over control) of the densitometric analysis of three different experiments in which either protein expression or mRNA expression were normalized to β-actin.

Abbreviations

BG

Bromocresol green

EGFR

Epidermal growth factor receptor

HIF-1α

Hypoxia-inducible factor-1α

MSK-1

Mitogen-and stress-activated kinase-1

PGE₂

Prostaglandin E₂

PGT

Prostaglandin transporter

VEGF-A

Vascular endothelial growth factor-A

RARβ

Retinoic acid receptor-β

RARE

Retinoic acid receptor response element