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Dual role of cAMP in the transcriptional regulation of multidrug resistance-associated protein 4 (MRP4) in pancreatic adenocarcinoma cell lines.

Carozzo A, Diez F, Gomez N, Cabrera M, Shayo C, Davio C, Fernández N - PLoS ONE (2015)

Bottom Line: Intracellular cAMP levels are tightly regulated where the MRP4 transporter plays a major role.Reporter luciferase experiments carried out in pancreatic AR42J cells showed that intracellular cAMP up-regulates MRP4 through an Epac2- and Rap1-mediated mechanism whereas extracellular cAMP reduced MRP4 promoter activity by a MEK/ERK-mediated pathway.Present results show that cAMP regulates MRP4 promoter activity, and further indicate that the balance between intracellular and extracellular cAMP levels determines MRP4 expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

ABSTRACT
Cyclic AMP represents one of the most studied signaling molecules and its role in proliferation and differentiation processes has been well established. Intracellular cAMP levels are tightly regulated where the MRP4 transporter plays a major role. In the present study, we sought to establish whether cAMP modulated MRP4 expression in pancreatic adenocarcinoma cell lines. Quantitative PCR and western blot studies showed that cAMP-increasing agents enhanced MRP4 transcripts and protein levels in PANC-1 cells. Reporter luciferase experiments carried out in pancreatic AR42J cells showed that intracellular cAMP up-regulates MRP4 through an Epac2- and Rap1-mediated mechanism whereas extracellular cAMP reduced MRP4 promoter activity by a MEK/ERK-mediated pathway. Present results show that cAMP regulates MRP4 promoter activity, and further indicate that the balance between intracellular and extracellular cAMP levels determines MRP4 expression.

No MeSH data available.


Related in: MedlinePlus

Effect of cAMP modulating agents on MRP4 expression in PANC-1 cells.Cells were exposed to agents that modulate the cAMP pathway at the indicated concentrations. Cells were harvested 24h after stimulation for mRNA quantitation, and after 48h for protein detection by immunoblotting. A. MRP4 mRNA was quantified by real-time PCR, normalized by β-actin mRNA and expressed relative to control (mean±SD; n = 3). B.top. Densitometric quantification of MRP4 protein bands normalized to β-actin and expressed relative to control (mean±SD; n = 6), bottom, representative western blot assay of six independent experiments is shown. *** p< 0.001.
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pone.0120651.g001: Effect of cAMP modulating agents on MRP4 expression in PANC-1 cells.Cells were exposed to agents that modulate the cAMP pathway at the indicated concentrations. Cells were harvested 24h after stimulation for mRNA quantitation, and after 48h for protein detection by immunoblotting. A. MRP4 mRNA was quantified by real-time PCR, normalized by β-actin mRNA and expressed relative to control (mean±SD; n = 3). B.top. Densitometric quantification of MRP4 protein bands normalized to β-actin and expressed relative to control (mean±SD; n = 6), bottom, representative western blot assay of six independent experiments is shown. *** p< 0.001.

Mentions: In order to evaluate the role of cAMP in modulating MRP4 expression in pancreatic cell lines, MRP4 mRNA transcripts were assessed by real-time PCR. PANC-1 cells, a widely used epithelial ductal pancreatic carcinoma model, were exposed to forskolin (adenylyl cyclase direct activator), IBMX (PDE inhibitor) and db-cAMP (cAMP permeable analog). Following cell exposure, all the agents assessed led to an approximate 2-fold increase in MRP4 mRNA levels as compared with untreated cells. These results indicate that cAMP increasing agents up-regulate MRP4. It is worth noting that cycloheximide treatment abolished db-cAMP-stimulated increase in MRP4 mRNA, suggesting that de novo synthesis is required for cAMP-mediated MRP4 transcription (Fig. 1A). In accordance, when MRP4 protein levels were analyzed by western blot, we observed a significant increase in MRP4 expression in cells exposed to forskolin, db-cAMP and IBMX (Fig. 1B). The increase in both mRNA and protein levels may result from a higher stability in mRNA transcripts as well as by MRP4 transcription stimulation. Therefore, we addressed MRP4 promoter activity by luciferase reporter assays.


Dual role of cAMP in the transcriptional regulation of multidrug resistance-associated protein 4 (MRP4) in pancreatic adenocarcinoma cell lines.

Carozzo A, Diez F, Gomez N, Cabrera M, Shayo C, Davio C, Fernández N - PLoS ONE (2015)

Effect of cAMP modulating agents on MRP4 expression in PANC-1 cells.Cells were exposed to agents that modulate the cAMP pathway at the indicated concentrations. Cells were harvested 24h after stimulation for mRNA quantitation, and after 48h for protein detection by immunoblotting. A. MRP4 mRNA was quantified by real-time PCR, normalized by β-actin mRNA and expressed relative to control (mean±SD; n = 3). B.top. Densitometric quantification of MRP4 protein bands normalized to β-actin and expressed relative to control (mean±SD; n = 6), bottom, representative western blot assay of six independent experiments is shown. *** p< 0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4366062&req=5

pone.0120651.g001: Effect of cAMP modulating agents on MRP4 expression in PANC-1 cells.Cells were exposed to agents that modulate the cAMP pathway at the indicated concentrations. Cells were harvested 24h after stimulation for mRNA quantitation, and after 48h for protein detection by immunoblotting. A. MRP4 mRNA was quantified by real-time PCR, normalized by β-actin mRNA and expressed relative to control (mean±SD; n = 3). B.top. Densitometric quantification of MRP4 protein bands normalized to β-actin and expressed relative to control (mean±SD; n = 6), bottom, representative western blot assay of six independent experiments is shown. *** p< 0.001.
Mentions: In order to evaluate the role of cAMP in modulating MRP4 expression in pancreatic cell lines, MRP4 mRNA transcripts were assessed by real-time PCR. PANC-1 cells, a widely used epithelial ductal pancreatic carcinoma model, were exposed to forskolin (adenylyl cyclase direct activator), IBMX (PDE inhibitor) and db-cAMP (cAMP permeable analog). Following cell exposure, all the agents assessed led to an approximate 2-fold increase in MRP4 mRNA levels as compared with untreated cells. These results indicate that cAMP increasing agents up-regulate MRP4. It is worth noting that cycloheximide treatment abolished db-cAMP-stimulated increase in MRP4 mRNA, suggesting that de novo synthesis is required for cAMP-mediated MRP4 transcription (Fig. 1A). In accordance, when MRP4 protein levels were analyzed by western blot, we observed a significant increase in MRP4 expression in cells exposed to forskolin, db-cAMP and IBMX (Fig. 1B). The increase in both mRNA and protein levels may result from a higher stability in mRNA transcripts as well as by MRP4 transcription stimulation. Therefore, we addressed MRP4 promoter activity by luciferase reporter assays.

Bottom Line: Intracellular cAMP levels are tightly regulated where the MRP4 transporter plays a major role.Reporter luciferase experiments carried out in pancreatic AR42J cells showed that intracellular cAMP up-regulates MRP4 through an Epac2- and Rap1-mediated mechanism whereas extracellular cAMP reduced MRP4 promoter activity by a MEK/ERK-mediated pathway.Present results show that cAMP regulates MRP4 promoter activity, and further indicate that the balance between intracellular and extracellular cAMP levels determines MRP4 expression.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio de Farmacología de Receptores, Cátedra de Química Medicinal, Departamento de Farmacología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina; Consejo Nacional de Investigaciones Científicas y Técnicas, Buenos Aires, Argentina.

ABSTRACT
Cyclic AMP represents one of the most studied signaling molecules and its role in proliferation and differentiation processes has been well established. Intracellular cAMP levels are tightly regulated where the MRP4 transporter plays a major role. In the present study, we sought to establish whether cAMP modulated MRP4 expression in pancreatic adenocarcinoma cell lines. Quantitative PCR and western blot studies showed that cAMP-increasing agents enhanced MRP4 transcripts and protein levels in PANC-1 cells. Reporter luciferase experiments carried out in pancreatic AR42J cells showed that intracellular cAMP up-regulates MRP4 through an Epac2- and Rap1-mediated mechanism whereas extracellular cAMP reduced MRP4 promoter activity by a MEK/ERK-mediated pathway. Present results show that cAMP regulates MRP4 promoter activity, and further indicate that the balance between intracellular and extracellular cAMP levels determines MRP4 expression.

No MeSH data available.


Related in: MedlinePlus