522 Rapid communication
cAMP regulates expression of the cyclic nucleotide transporter MRP4 (ABCC4) through the EPAC pathway Susanne Bröderdorf, Sebastian Zang, Yvonne Schaletzki, Markus Grube, Heyo K. Kroemer and Gabriele Jedlitschky Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate
Introduction Cyclic AMP (cAMP) is one of the oldest known signaling molecules. The classical cAMP target, the cAMPdependent protein kinase (PKA), subsequently phosphorylates downstream effectors such as transcription factors of the cyclic AMP-responsive element binding protein (CREB) family [1]. In addition to PKA, recent evidence has highlighted a major role for guaninenucleotide exchange factors for Rap proteins in mediating cAMP signaling, namely, the exchange proteins directly activated by cyclic AMP (EPAC) with the two mammalian gene variants EPAC1 and EPAC2 [2]. cAMP influences diverse cellular processes by EPAC signaling including regulation of ion channel function, transporter activity, and exocytosis in multiple tissues. Downstream targets of EPAC include the mitogen-activated protein/ extracellular signal-regulated kinase (MAPK/ERK) kinase (MEK) cascade [2]. Phosphatidylinositol 3′-kinase (PI3K) and p38 represent additional cAMP targets. Classically, the regulation of cAMP signaling involves the degradation by phosphodiesterases (PDEs). However, cyclic nucleotide efflux transporters of the multidrug resistance protein (MRP) family, which is part of the ATP-binding cassette (ABC) transporter subfamily C, Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.pharmacogeneticsandgenomics.com). 1744-6872 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
MRP4 expression, which can result in increased cAMP efflux. Pharmacogenetics and Genomics 24:522–526 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:522–526 Keywords: cyclic AMP, exchange protein activated by cyclic AMP, mitogen-activated protein kinases, multidrug resistance protein 4 (MRP4/ABCC4), reporter gene assay, transcriptional regulation Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany Correspondence to Gabriele Jedlitschky, PhD, Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine, Felix-Hausdorff Street 3, D-17487 Greifswald, Germany Tel: + 49 3834 8622146; fax: + 49 3834 865631; e-mail: [email protected] Present address: Heyo K. Kroemer: University Medicine Göttingen, Germany. Received 18 December 2013 Accepted 18 July 2014
have also been implicated in the regulation of cyclic nucleotide signaling [3,4]. Cyclic nucleotides have been identified as substrates for MRP4 (ABCC4), MRP5 (ABCC5), and MRP8 (ABCC11) [5–7]. Especially MRP4 has been established as an independent regulator of cAMP-mediated signaling pathways in particular in vascular smooth muscle cells (SMC) as well as in hematopoietic cells [8,9]. An upregulation of several PDEs by cAMP-stimulating agents is known to be part of an autoregulatory positive feedback mechanism. However, little is known so far on the regulation of MRP4. Therefore, we studied the effect of long-term treatment with a cell-permeable cAMP analog and PDE inhibition on the expression of MRP4 in different cell types as well as the signaling pathway leading to the observed gene regulation.
Materials and methods Cell experiments
HeLa cells and the human megakaryoblastic leukemia M07e cells [10] were grown in RPMI-1640 medium (10% fetal calf serum, 2 mmol/l L-glutamine). SMC isolated from the human coronary artery (PromoCell, Heidelberg, Germany) were cultured in smooth muscle cell growth medium 2 (PromoCell). For induction experiments, the cells were incubated at 37°C with dibutyryl cAMP (dbcAMP; Sigma, St. Louis, Missouri, USA), 3-isobutylDOI: 10.1097/FPC.0000000000000084
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Regulation of MRP4 by cAMP Bröderdorf et al. 523
1-methylxanthine (IBMX; Sigma), and 8-(4-chloro-phenylthio)-2-O-methyl cAMP (8-CPT-Me-cAMP; Tocris, Bristol, UK) or with the respective solvent (dilution of dimethyl sulfoxide) as controls as presented in detail in the figure legends. Inhibitors such as KT5720 and brefeldin A (Sigma), PD98059, wortmannin and SB203580 (Calbiochem, Darmstadt, Germany), and apigenin (Sigma) were added 30 min before db-cAMP treatment. For EPAC silencing, M07e cells were transfected with EPAC siRNA or control siRNA (sc-41700 and sc-37007; Santa Cruz Biotechnology, Santa Cruz, California, USA) using lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, California, USA) 48 h before treatment. Quantitative real-time PCR
Total RNA was isolated from the cells using PeqGold RNAPure (Peqlab, Erlangen, Germany) and transcribed (500 ng RNA/20 μl reaction volume). Real-time PCR was performed using the ABI prism 7900 sequence detection system and assays on demand for ABCC4 (Hs00988717_m1) conjugated with fluorochrome 5-carboxyfluorescein, 18S rRNA (predeveloped TaqMan assay reagent) conjugated with fluorochrome FAM, and TaqMan gene expression master mix (Applied Biosystems, Foster City, California, USA). Quantification was performed using the comparative ΔΔCt method. Dual-luciferase reporter gene assay
A 1494-bp fragment of the 5′ upstream region of the MRP4/ABCC4 gene (see Supplemental digital content 1, http://links.lww.com/FPC/A764) was amplified and cloned into the pGL3-basic firefly luciferase reporter vector (Promega, Mannheim, Germany), and the correct sequence was analyzed by sequencing. HeLa cells were transfected with the reporter construct and treated for 24 h with the compounds presented in Fig. 1. Each experiment was conducted by cotransfection of the pRLTK Renilla luciferase vector, which was used for normalization. After cell lysis, luminescent signals generated by the dual-luciferase reporter assay system (Promega) were detected and data were calculated as the ratio of firefly/Renilla luminescence. Immunoblot and immunofluorescence microscopy
Immunoblots and immunofluorescence microscopy were performed as described before [10,11]. For detection of MRP4, the rabbit antiserum SNG [11] (kindly provided by Dr Dietrich Keppler, DKFZ, Germany) was used. The other primary antibodies were anti-EPAC (A-5), anti-β-actin (Santa Cruz Biotechnology), and anti-pVASP (Ser-157), anti-VASP, anti-pERK, and anti-ERK (Cell Signaling Technology, Danvers, Massachusetts, USA). Alexa Fluor 488 secondary antibody for immunofluorescence was from Molecular Probes (Eugene, Oregon, USA). Cell surface biotinylation was performed using sulfo-NHS-SS-biotin as described before [12]. After fixation of the cells, biotinylated surface proteins
were stained using fluorescein isothiocyanate-labeled streptavidin (ThermoFisher Scientific, Rockford, Illinois, USA). Statistical analysis
Data are presented as the mean ± SD and differences between groups were statistically analyzed using Student’s t-test or one-way analysis of variance as appropriate and GraphPad Prism 5.01 software (GraphPad, San Diego, California, USA).
Results and discussion Transcriptional regulation of MRP4 in HeLa cells
To test the effect of long-term increased intracellular cAMP concentrations on MRP4 expression, we treated HeLa cells (as a model cell line expressing MRP4) with the cell-permeable cAMP analog db-cAMP or with the unspecific PDE inhibitor IBMX and analyzed MRP4 expression at the mRNA and protein level 24 and 48 h after the addition of db-cAMP, respectively. As shown in Fig. 1a and b, MRP4 protein and mRNA were significantly increased after the addition of db-cAMP. In these cells, both db-cAMP and IBMX alone were sufficient to upregulate the MRP4 protein. Furthermore, we transfected HeLa cells with a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene fused to the firefly luciferase reporter gene. After the addition of db-cAMP (for 24 h), luciferase activity was increased about three-fold compared with the basic activity (Fig. 1c). Similar results were obtained when the data were normalized to Renilla control vectors containing different promoters (HSV-thymidine kinase, CMV, SV40), indicating that the effect is not because of possible cAMP effects on the internal control. To unravel the signaling pathways involved, inhibitors of several kinases were added. Pretreatment of the cells with the MAPK/ERK inhibitor PD98059 [13] completely repressed the db-cAMP-induced activity increase, whereas no statistically significant effects were observed with the PKA inhibitor KT5720 [13] as well as wortmannin and SB203580, which were used as inhibitors of PI3K and p38, respectively [13] (Fig. 1c). PD98059 also reverted the db-cAMP-induced increase in MRP4 protein and mRNA (Fig. 1a and b). The MRP4 promoter contains two putative CREB-binding sites (at positions − 173 and − 810) as targets of PKA signaling; however, mutation of these sites in the reporter plasmid did not abolish the cAMP-mediated induction. The increase in luciferase activity by db-cAMP (246 ± 13 vs. 100 ± 8% control; n = 5) obtained with the mutated plasmid did not differ significantly from the one obtained with the wildtype promoter construct (268 ± 23 vs. 100 ± 8%; n = 5). These results suggested that the signal is rather transducted by the EPAC/MEK route than by the PKA/CREB pathway. To confirm that both pathways were active and affected by the respective inhibitors,
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524 Pharmacogenetics and Genomics 2014, Vol 24 No 10
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cAMP regulates MRP4 expression in HeLa cells. (a, b) Cells were treated with 1 mmol/l db-cAMP or 200 μmol/l IBMX or 1 mmol/l db-cAMP in the presence of the MAPK/ERK inhibitor PD98059 (25 μmol/l) (+ PD98059). The cells were harvested 24 h after the addition of db-cAMP for mRNA isolation and after 48 h for protein detection by immunoblotting. (a) Densitometric quantification of MRP4 protein bands normalized to β-actin and related to the unstimulated control is presented (mean ± SD; n = 3). (b) MRP4 mRNA was quantified by real-time PCR (normalized to 18S rRNA) and related to the untreated control (mean ± SD; n = 6). (c) Cells were transfected with the pGL3 luciferase reporter vector containing a 1494-bp fragment of the 5′ regulatory region of MRP4/ABCC4 and treated with 1 mmol/l db-cAMP or 1 mmol/l db-cAMP combined with the inhibitors KT5720 (10 μmol/l), PD98059 (25 μmol/l), wortmannin (100 nmol/l), and SB203580 (10 μmol/l). Firefly luciferase activities were measured after 24 h in the cell lysates and normalized to Renilla luciferase (cotransfection with pRL-TK) (mean ± SD; unstimulated control = 1; n = 5). (d, e) Cells were stimulated again with db-cAMP and IBMX (100 μmol/l each) and cotreated with KT5720 (10 μmol/l) or PD98059 (25 μmol/l). In addition to MRP4 protein, which was detected as described above, pERK and total ERK as well as VASP phosphorylated at Ser-157 (pVASP) and total VASP were analyzed by immunoblotting (24 h after the addition of db-cAMP). Blots (d) and quantitative evaluation of MRP4/β-actin, pERK/ERK, and pVASP/VASP ratios (e) are presented from one representative of three similar experiments. Statistically significant differences with **P < 0.01, ***P < 0.001. ABC, ATP-binding cassette; cAMP, cyclic AMP; db-cAMP, dibutyryl cyclic AMP; ERK, extracellular signal-regulated kinase; IBMX, 3-isobutyl-1-methylxanthine; MRP4, multidrug resistance protein 4; pERK, phosphorylated ERK.
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Regulation of MRP4 by cAMP Bröderdorf et al. 525
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Upregulation of MRP4 in vascular SMC and myeloid leukemia cells (M07e) through EPAC signaling. (a) Detection of MRP4 and of EPAC in SMC and M07e cells by immunoblotting. (b) MRP4 localization in SMC (upper panels) and in M07e cells (lower panels): cells were treated with solvent (Co) or with the specific EPAC activator 8-CPT-Me-cAMP (8-CPT; 300 μmol/l; 24 h) and cell surface proteins were subsequently biotinylated. After fixation, cells were probed with the MRP4 antibody (red fluorescence) and fluorescein isothiocyanate-labeled streptavidin (green). Yellow fluorescence indicates colocalized pixels. Fluorescence micrographs were taken using a confocal laser scanning microscope (Zeiss LSM 780) and evaluated for colocalization using the ZEN software (Zeiss, Jena, Germany) (blue: DAPI staining of nuclei; scale bars = 10 μm). (c, d) MRP4 mRNA was quantified by real-time PCR 24 h after the addition of the stimulating agents, normalized to 18S rRNA, and related to the unstimulated control. SMC (c) and M07e cells (d) were stimulated with 100 μmol/l db-cAMP (+ 100 μmol/l IBMX) or with db-cAMP + IBMX in the presence of the PKA inhibitor KT5720 (10 μmol/l) (left panels) or in the presence of brefeldin A and apigenin (100 μmol/l each) (shown only for SMC). In addition, cells were stimulated with 8-CPT (300 μmol/l) (shown for SMC in the right panel; for M07e cells in the left panel). (d) Left panel: M07e cells were transfected with EPAC siRNA or control siRNA 48 h before the addition of db-cAMP (+ IBMX) and 8-CPT (mean ± SD; n = 6). The effective knockdown of EPAC protein by the EPAC siRNA treatment was confirmed by immunoblotting (not shown), which showed a decrease in the EPAC/β-actin ratio of ∼ 50% after 48 h. Statistically significant differences with *P < 0.05, **P < 0.01, and ***P < 0.001. cAMP, cyclic AMP; db-cAMP, dibutyryl cyclic AMP; EPAC, exchange proteins activated by cyclic AMP; IBMX, 3-isobutyl-1-methylxanthine; MRP4, multidrug resistance protein 4; SMC, smooth muscle cell.
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526 Pharmacogenetics and Genomics 2014, Vol 24 No 10
phosphorylation of VASP at Ser-157 (positive control for PKA) and phosphorylation of ERK (EPAC pathway) were detected in parallel (Fig. 1d and e). Regulation of MRP4 in myeloid leukemia cells and vascular SMC
In addition, we used the M07e myeloid leukemia cells [10] and vascular SMC. Both cell lines express MRP4 and EPAC (Fig. 2a). In SMC MRP4 staining was observed at the plasma membrane (labeled with biotin in Fig. 2b) and in intracellular structures preferentially near the nuclei, which may represent staining of MRP4 protein that is processed in the Golgi system. The relative cell surface expression of MRP4 was unchanged after its induction by 8-CPT-Me-cAMP (8-CPT), a selective EPAC activator [14]. This is indicated by similar intensity-weighted colocalization coefficients of MRP4 pixels that colocalized with biotin pixels amounting to 27 ± 7 and 32 ± 12% in control and 8-CPT-stimulated cells, respectively (n = 6 evaluated images). In contrast, in M07e cells, MRP4 was mainly detected intracellularly before and after the induction (Fig. 2b). Stimulation with db-cAMP produced a significant increase in MRP4 mRNA levels also in these cells (Fig. 2c and d), but only in the presence of IBMX. This may be because of the high PDE activities in these cell lines. Again, the effect could not be reverted by the addition of the PKA inhibitor KT5720. However, a significant effect was observed for brefeldin A and apigenin, which have been described as inhibitors (albeit rather unspecific) of EPAC signaling and MAPKs, respectively [14,15]. In addition, 8-CPT could induce MRP4 transcription to a similar or even higher extent than db-cAMP (+ IBMX). This upregulation by 8-CPT was also observed at the protein level with a 1.5-fold and two-fold increase in the M07e cells and SMC, respectively. Under the experimental conditions used, cell proliferation was found to be unchanged or slightly decreased (see Fig. 2 of Supplemental digital content 1, http://links.lww.com/ FPC/A764). To further confirm the hypothesis that the EPAC/MEK pathway is involved in this regulation, M07e cells were transfected with EPAC and control siRNA and subsequently stimulated with db-cAMP and 8-CPT. In contrast to the control siRNA, the pretreatment with EPAC siRNA significantly reduced the induction of MRP4 (Fig. 2d, right panel). Conclusion
Our results suggest that long-term treatment with cAMPenhancing agents may lead to an increased MRP4 expression as part of an autoregulatory positive feedback loop. As a result, efflux or intracellular sequestration of cAMP and other substrates may be increased (depending on the subcellular localization of MRP4). This regulatory mechanism may also counteract the effect of long-term treatment with cAMP-enhancing drugs as β-adrenergic agonists or PDE inhibitors.
Acknowledgements The authors thank Dr Igor Mosyagin, Department of Pharmacology, University Medicine Greifswald, for his helpful suggestions and discussions. The antiserum SNG was kindly provided by Dr Dietrich Keppler, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany. The work was supported by the Deutsche Forschung sgemeinschaft (DFG) through grant JE 234/4-1 to G.J. and by the European Union through grant EU-FP7REGPOT-2010-1 (‘EnVision’, grant agreement no. 264143). Conflicts of interest
There are no conflicts of interest.
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cAMP regulates expression of the cyclic nucleotide transporter MRP4 (ABCC4) through the EPAC pathway Susanne Bröderdorf, Sebastian Zang, Yvonne Schaletzki, Markus Grube, Heyo K. Kroemer and Gabriele Jedlitschky Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate
Introduction Cyclic AMP (cAMP) is one of the oldest known signaling molecules. The classical cAMP target, the cAMPdependent protein kinase (PKA), subsequently phosphorylates downstream effectors such as transcription factors of the cyclic AMP-responsive element binding protein (CREB) family [1]. In addition to PKA, recent evidence has highlighted a major role for guaninenucleotide exchange factors for Rap proteins in mediating cAMP signaling, namely, the exchange proteins directly activated by cyclic AMP (EPAC) with the two mammalian gene variants EPAC1 and EPAC2 [2]. cAMP influences diverse cellular processes by EPAC signaling including regulation of ion channel function, transporter activity, and exocytosis in multiple tissues. Downstream targets of EPAC include the mitogen-activated protein/ extracellular signal-regulated kinase (MAPK/ERK) kinase (MEK) cascade [2]. Phosphatidylinositol 3′-kinase (PI3K) and p38 represent additional cAMP targets. Classically, the regulation of cAMP signaling involves the degradation by phosphodiesterases (PDEs). However, cyclic nucleotide efflux transporters of the multidrug resistance protein (MRP) family, which is part of the ATP-binding cassette (ABC) transporter subfamily C, Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.pharmacogeneticsandgenomics.com). 1744-6872 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
MRP4 expression, which can result in increased cAMP efflux. Pharmacogenetics and Genomics 24:522–526 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:522–526 Keywords: cyclic AMP, exchange protein activated by cyclic AMP, mitogen-activated protein kinases, multidrug resistance protein 4 (MRP4/ABCC4), reporter gene assay, transcriptional regulation Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine, Greifswald, Germany Correspondence to Gabriele Jedlitschky, PhD, Department of Pharmacology, Center of Drug Absorption and Transport (C_DAT), University Medicine, Felix-Hausdorff Street 3, D-17487 Greifswald, Germany Tel: + 49 3834 8622146; fax: + 49 3834 865631; e-mail: [email protected] Present address: Heyo K. Kroemer: University Medicine Göttingen, Germany. Received 18 December 2013 Accepted 18 July 2014
have also been implicated in the regulation of cyclic nucleotide signaling [3,4]. Cyclic nucleotides have been identified as substrates for MRP4 (ABCC4), MRP5 (ABCC5), and MRP8 (ABCC11) [5–7]. Especially MRP4 has been established as an independent regulator of cAMP-mediated signaling pathways in particular in vascular smooth muscle cells (SMC) as well as in hematopoietic cells [8,9]. An upregulation of several PDEs by cAMP-stimulating agents is known to be part of an autoregulatory positive feedback mechanism. However, little is known so far on the regulation of MRP4. Therefore, we studied the effect of long-term treatment with a cell-permeable cAMP analog and PDE inhibition on the expression of MRP4 in different cell types as well as the signaling pathway leading to the observed gene regulation.
Materials and methods Cell experiments
HeLa cells and the human megakaryoblastic leukemia M07e cells [10] were grown in RPMI-1640 medium (10% fetal calf serum, 2 mmol/l L-glutamine). SMC isolated from the human coronary artery (PromoCell, Heidelberg, Germany) were cultured in smooth muscle cell growth medium 2 (PromoCell). For induction experiments, the cells were incubated at 37°C with dibutyryl cAMP (dbcAMP; Sigma, St. Louis, Missouri, USA), 3-isobutylDOI: 10.1097/FPC.0000000000000084
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
Regulation of MRP4 by cAMP Bröderdorf et al. 523
1-methylxanthine (IBMX; Sigma), and 8-(4-chloro-phenylthio)-2-O-methyl cAMP (8-CPT-Me-cAMP; Tocris, Bristol, UK) or with the respective solvent (dilution of dimethyl sulfoxide) as controls as presented in detail in the figure legends. Inhibitors such as KT5720 and brefeldin A (Sigma), PD98059, wortmannin and SB203580 (Calbiochem, Darmstadt, Germany), and apigenin (Sigma) were added 30 min before db-cAMP treatment. For EPAC silencing, M07e cells were transfected with EPAC siRNA or control siRNA (sc-41700 and sc-37007; Santa Cruz Biotechnology, Santa Cruz, California, USA) using lipofectamine RNAiMAX reagent (Life Technologies, Carlsbad, California, USA) 48 h before treatment. Quantitative real-time PCR
Total RNA was isolated from the cells using PeqGold RNAPure (Peqlab, Erlangen, Germany) and transcribed (500 ng RNA/20 μl reaction volume). Real-time PCR was performed using the ABI prism 7900 sequence detection system and assays on demand for ABCC4 (Hs00988717_m1) conjugated with fluorochrome 5-carboxyfluorescein, 18S rRNA (predeveloped TaqMan assay reagent) conjugated with fluorochrome FAM, and TaqMan gene expression master mix (Applied Biosystems, Foster City, California, USA). Quantification was performed using the comparative ΔΔCt method. Dual-luciferase reporter gene assay
A 1494-bp fragment of the 5′ upstream region of the MRP4/ABCC4 gene (see Supplemental digital content 1, http://links.lww.com/FPC/A764) was amplified and cloned into the pGL3-basic firefly luciferase reporter vector (Promega, Mannheim, Germany), and the correct sequence was analyzed by sequencing. HeLa cells were transfected with the reporter construct and treated for 24 h with the compounds presented in Fig. 1. Each experiment was conducted by cotransfection of the pRLTK Renilla luciferase vector, which was used for normalization. After cell lysis, luminescent signals generated by the dual-luciferase reporter assay system (Promega) were detected and data were calculated as the ratio of firefly/Renilla luminescence. Immunoblot and immunofluorescence microscopy
Immunoblots and immunofluorescence microscopy were performed as described before [10,11]. For detection of MRP4, the rabbit antiserum SNG [11] (kindly provided by Dr Dietrich Keppler, DKFZ, Germany) was used. The other primary antibodies were anti-EPAC (A-5), anti-β-actin (Santa Cruz Biotechnology), and anti-pVASP (Ser-157), anti-VASP, anti-pERK, and anti-ERK (Cell Signaling Technology, Danvers, Massachusetts, USA). Alexa Fluor 488 secondary antibody for immunofluorescence was from Molecular Probes (Eugene, Oregon, USA). Cell surface biotinylation was performed using sulfo-NHS-SS-biotin as described before [12]. After fixation of the cells, biotinylated surface proteins
were stained using fluorescein isothiocyanate-labeled streptavidin (ThermoFisher Scientific, Rockford, Illinois, USA). Statistical analysis
Data are presented as the mean ± SD and differences between groups were statistically analyzed using Student’s t-test or one-way analysis of variance as appropriate and GraphPad Prism 5.01 software (GraphPad, San Diego, California, USA).
Results and discussion Transcriptional regulation of MRP4 in HeLa cells
To test the effect of long-term increased intracellular cAMP concentrations on MRP4 expression, we treated HeLa cells (as a model cell line expressing MRP4) with the cell-permeable cAMP analog db-cAMP or with the unspecific PDE inhibitor IBMX and analyzed MRP4 expression at the mRNA and protein level 24 and 48 h after the addition of db-cAMP, respectively. As shown in Fig. 1a and b, MRP4 protein and mRNA were significantly increased after the addition of db-cAMP. In these cells, both db-cAMP and IBMX alone were sufficient to upregulate the MRP4 protein. Furthermore, we transfected HeLa cells with a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene fused to the firefly luciferase reporter gene. After the addition of db-cAMP (for 24 h), luciferase activity was increased about three-fold compared with the basic activity (Fig. 1c). Similar results were obtained when the data were normalized to Renilla control vectors containing different promoters (HSV-thymidine kinase, CMV, SV40), indicating that the effect is not because of possible cAMP effects on the internal control. To unravel the signaling pathways involved, inhibitors of several kinases were added. Pretreatment of the cells with the MAPK/ERK inhibitor PD98059 [13] completely repressed the db-cAMP-induced activity increase, whereas no statistically significant effects were observed with the PKA inhibitor KT5720 [13] as well as wortmannin and SB203580, which were used as inhibitors of PI3K and p38, respectively [13] (Fig. 1c). PD98059 also reverted the db-cAMP-induced increase in MRP4 protein and mRNA (Fig. 1a and b). The MRP4 promoter contains two putative CREB-binding sites (at positions − 173 and − 810) as targets of PKA signaling; however, mutation of these sites in the reporter plasmid did not abolish the cAMP-mediated induction. The increase in luciferase activity by db-cAMP (246 ± 13 vs. 100 ± 8% control; n = 5) obtained with the mutated plasmid did not differ significantly from the one obtained with the wildtype promoter construct (268 ± 23 vs. 100 ± 8%; n = 5). These results suggested that the signal is rather transducted by the EPAC/MEK route than by the PKA/CREB pathway. To confirm that both pathways were active and affected by the respective inhibitors,
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524 Pharmacogenetics and Genomics 2014, Vol 24 No 10
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cAMP regulates MRP4 expression in HeLa cells. (a, b) Cells were treated with 1 mmol/l db-cAMP or 200 μmol/l IBMX or 1 mmol/l db-cAMP in the presence of the MAPK/ERK inhibitor PD98059 (25 μmol/l) (+ PD98059). The cells were harvested 24 h after the addition of db-cAMP for mRNA isolation and after 48 h for protein detection by immunoblotting. (a) Densitometric quantification of MRP4 protein bands normalized to β-actin and related to the unstimulated control is presented (mean ± SD; n = 3). (b) MRP4 mRNA was quantified by real-time PCR (normalized to 18S rRNA) and related to the untreated control (mean ± SD; n = 6). (c) Cells were transfected with the pGL3 luciferase reporter vector containing a 1494-bp fragment of the 5′ regulatory region of MRP4/ABCC4 and treated with 1 mmol/l db-cAMP or 1 mmol/l db-cAMP combined with the inhibitors KT5720 (10 μmol/l), PD98059 (25 μmol/l), wortmannin (100 nmol/l), and SB203580 (10 μmol/l). Firefly luciferase activities were measured after 24 h in the cell lysates and normalized to Renilla luciferase (cotransfection with pRL-TK) (mean ± SD; unstimulated control = 1; n = 5). (d, e) Cells were stimulated again with db-cAMP and IBMX (100 μmol/l each) and cotreated with KT5720 (10 μmol/l) or PD98059 (25 μmol/l). In addition to MRP4 protein, which was detected as described above, pERK and total ERK as well as VASP phosphorylated at Ser-157 (pVASP) and total VASP were analyzed by immunoblotting (24 h after the addition of db-cAMP). Blots (d) and quantitative evaluation of MRP4/β-actin, pERK/ERK, and pVASP/VASP ratios (e) are presented from one representative of three similar experiments. Statistically significant differences with **P < 0.01, ***P < 0.001. ABC, ATP-binding cassette; cAMP, cyclic AMP; db-cAMP, dibutyryl cyclic AMP; ERK, extracellular signal-regulated kinase; IBMX, 3-isobutyl-1-methylxanthine; MRP4, multidrug resistance protein 4; pERK, phosphorylated ERK.
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Regulation of MRP4 by cAMP Bröderdorf et al. 525
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Upregulation of MRP4 in vascular SMC and myeloid leukemia cells (M07e) through EPAC signaling. (a) Detection of MRP4 and of EPAC in SMC and M07e cells by immunoblotting. (b) MRP4 localization in SMC (upper panels) and in M07e cells (lower panels): cells were treated with solvent (Co) or with the specific EPAC activator 8-CPT-Me-cAMP (8-CPT; 300 μmol/l; 24 h) and cell surface proteins were subsequently biotinylated. After fixation, cells were probed with the MRP4 antibody (red fluorescence) and fluorescein isothiocyanate-labeled streptavidin (green). Yellow fluorescence indicates colocalized pixels. Fluorescence micrographs were taken using a confocal laser scanning microscope (Zeiss LSM 780) and evaluated for colocalization using the ZEN software (Zeiss, Jena, Germany) (blue: DAPI staining of nuclei; scale bars = 10 μm). (c, d) MRP4 mRNA was quantified by real-time PCR 24 h after the addition of the stimulating agents, normalized to 18S rRNA, and related to the unstimulated control. SMC (c) and M07e cells (d) were stimulated with 100 μmol/l db-cAMP (+ 100 μmol/l IBMX) or with db-cAMP + IBMX in the presence of the PKA inhibitor KT5720 (10 μmol/l) (left panels) or in the presence of brefeldin A and apigenin (100 μmol/l each) (shown only for SMC). In addition, cells were stimulated with 8-CPT (300 μmol/l) (shown for SMC in the right panel; for M07e cells in the left panel). (d) Left panel: M07e cells were transfected with EPAC siRNA or control siRNA 48 h before the addition of db-cAMP (+ IBMX) and 8-CPT (mean ± SD; n = 6). The effective knockdown of EPAC protein by the EPAC siRNA treatment was confirmed by immunoblotting (not shown), which showed a decrease in the EPAC/β-actin ratio of ∼ 50% after 48 h. Statistically significant differences with *P < 0.05, **P < 0.01, and ***P < 0.001. cAMP, cyclic AMP; db-cAMP, dibutyryl cyclic AMP; EPAC, exchange proteins activated by cyclic AMP; IBMX, 3-isobutyl-1-methylxanthine; MRP4, multidrug resistance protein 4; SMC, smooth muscle cell.
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526 Pharmacogenetics and Genomics 2014, Vol 24 No 10
phosphorylation of VASP at Ser-157 (positive control for PKA) and phosphorylation of ERK (EPAC pathway) were detected in parallel (Fig. 1d and e). Regulation of MRP4 in myeloid leukemia cells and vascular SMC
In addition, we used the M07e myeloid leukemia cells [10] and vascular SMC. Both cell lines express MRP4 and EPAC (Fig. 2a). In SMC MRP4 staining was observed at the plasma membrane (labeled with biotin in Fig. 2b) and in intracellular structures preferentially near the nuclei, which may represent staining of MRP4 protein that is processed in the Golgi system. The relative cell surface expression of MRP4 was unchanged after its induction by 8-CPT-Me-cAMP (8-CPT), a selective EPAC activator [14]. This is indicated by similar intensity-weighted colocalization coefficients of MRP4 pixels that colocalized with biotin pixels amounting to 27 ± 7 and 32 ± 12% in control and 8-CPT-stimulated cells, respectively (n = 6 evaluated images). In contrast, in M07e cells, MRP4 was mainly detected intracellularly before and after the induction (Fig. 2b). Stimulation with db-cAMP produced a significant increase in MRP4 mRNA levels also in these cells (Fig. 2c and d), but only in the presence of IBMX. This may be because of the high PDE activities in these cell lines. Again, the effect could not be reverted by the addition of the PKA inhibitor KT5720. However, a significant effect was observed for brefeldin A and apigenin, which have been described as inhibitors (albeit rather unspecific) of EPAC signaling and MAPKs, respectively [14,15]. In addition, 8-CPT could induce MRP4 transcription to a similar or even higher extent than db-cAMP (+ IBMX). This upregulation by 8-CPT was also observed at the protein level with a 1.5-fold and two-fold increase in the M07e cells and SMC, respectively. Under the experimental conditions used, cell proliferation was found to be unchanged or slightly decreased (see Fig. 2 of Supplemental digital content 1, http://links.lww.com/ FPC/A764). To further confirm the hypothesis that the EPAC/MEK pathway is involved in this regulation, M07e cells were transfected with EPAC and control siRNA and subsequently stimulated with db-cAMP and 8-CPT. In contrast to the control siRNA, the pretreatment with EPAC siRNA significantly reduced the induction of MRP4 (Fig. 2d, right panel). Conclusion
Our results suggest that long-term treatment with cAMPenhancing agents may lead to an increased MRP4 expression as part of an autoregulatory positive feedback loop. As a result, efflux or intracellular sequestration of cAMP and other substrates may be increased (depending on the subcellular localization of MRP4). This regulatory mechanism may also counteract the effect of long-term treatment with cAMP-enhancing drugs as β-adrenergic agonists or PDE inhibitors.
Acknowledgements The authors thank Dr Igor Mosyagin, Department of Pharmacology, University Medicine Greifswald, for his helpful suggestions and discussions. The antiserum SNG was kindly provided by Dr Dietrich Keppler, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, Germany. The work was supported by the Deutsche Forschung sgemeinschaft (DFG) through grant JE 234/4-1 to G.J. and by the European Union through grant EU-FP7REGPOT-2010-1 (‘EnVision’, grant agreement no. 264143). Conflicts of interest
There are no conflicts of interest.
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