Influence of Phosphodiesterase Inhibition on CRE- and EGR1-Dependent Transcription in a Mouse Hippocampal Cell Line.

Erik Maronde

doi:10.3390/ijms21228658

Abstract

Signaling pathways, depending on the second messenger molecule cAMP, modulate hippocampal cell signaling via influencing transcription factors like cAMP-regulated element-binding protein (CREB) or early growth response 1 EGR1/Krox24/zif268/ZENK (EGR1). Here, we investigated two reporter cell lines derived from an immortalized hippocampal neuronal cell line stably expressing a CRE- or EGR1-luciferase reporter gene (HT22CREluc and HT22EGR1luc, respectively). The cells were subjected to phosphodiesterase inhibitors and other cAMP-modulating agents to investigate dose- and time-dependent phosphodiesterase (PDE)-mediated fine-tuning of cAMP-dependent transcriptional signaling. The non-isoform-specific cyclic nucleotide phosphodiesterase (PDE) inhibitor isobutyl-methyl-xanthine (IBMX), as well as selective inhibitors of PDE3 (milrinone) and PDE4 (rolipram), were tested for their ability to elevate CRE- and EGR1-luciferase activity. Pharmacological parameters like onset of activity, maximum activity, and offset of activity were determined. In summary, phosphodiesterase inhibition appeared similarly potent in comparison to adenylate cyclase stimulation or direct activation of protein kinase A (PKA) via specific cAMP agonists and was at least partly mediated by PKA as shown by the selective PKA inhibitor Rp-8-Br-cAMPS. Moreover, transcriptional activation by PDE inhibition was also influenced by organic anion-exchanger action and interacted with fibroblast growth factor (FGF) receptor-mediated pathways.

Highlights

Intracellular levels of cAMP are regulated by substances activating or inhibiting the enzyme adenylate cyclase (AC), which catalyzes the reaction of ATP to cAMP [1]
Elevated levels of cAMP lead to binding to various effector proteins like cAMP-dependent protein kinase A (PKA), exchange factor directly activated by cAMP (EPAC), or different kinds of cyclic nucleotide-gated (CNG) ion channels [2,3,4,5]
We investigated the interaction of rolipram with fibroblast growth factor 1 (FGF1)-stimulated fibroblast growth factor (FGF) receptor tyrosine phosphorylation in order to evaluate the interaction of the PDE/cAMP/PKA/cAMP-regulated element-binding protein (CREB) pathway with another prominent cell signaling pathway involved in hippocampal function

Summary

Introduction

Intracellular levels of cAMP are regulated by substances activating or inhibiting the enzyme adenylate cyclase (AC), which catalyzes the reaction of ATP to cAMP [1]. The degradation of cAMP is accomplished by the enzyme cyclic nucleotide phosphodiesterase (PDE), of which eleven isozyme families have been described so far [6]. Through the regulation of cAMP levels, PDEs are involved in synaptic plasticity processes such as those observed during memory processes, aging, and neurodegeneration [6,7,8]. Transcriptional processes regulated by the second messenger 3’,5’-adenosine monophosphate (cAMP) are important in these processes [11]. The upregulation of PDE4 during sleep deprivation has been described to lead to memory deficits, which were compensated by application of the PDE4-specific inhibitor rolipram [13]. Whether inhibition of PDE is sufficient for the activation of CREand EGR1-dependent transcriptional pathways similar to AC activation is not clear and will be investigated here

Methods

Results

Conclusion