Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors

Molecular mechanisms underlying PDE3A-caused hypertension with brachydactyly (HTNB)

Hypertension represents the main risk factor for cardiovascular death worldwide. Autosomal dominant hypertension with brachydactyly (HTNB) is a salt- resistant Mendelian syndrome characterized by progressive hypertension, brachydactyly type E and blood vessel hyperplasia. It resembles essential hypertension. If untreated, patients die from stroke before age 50 years. This study provides strong evidence that mutations affecting a 15 bp regulatory region of the phosphodiesterase 3A (PDE3A) gene cause HTNB. A new human mutation that affects the 15 bp region of the PDE3A gene was identified. A CRISPR/Cas9 rat model was generated carrying a 9 bp deletion in the same regulatory region, which is analogous to a human 3 bp deletion and which recapitulates HTNB. The mutated PDE3A gene drives mechanisms that increase vascular smooth muscle cell (VSMC) proliferation and peripheral vascular resistance causing hypertension; heart function, however, appears normal. PDE3A hydrolyses cyclic adenosine-3’,5’-monophosphate (cAMP) and is inhibited by cyclic guanosine- 3’,5’-monophosphate (cGMP). Treatment of the mutant rats with the soluble guanylyl cyclase (sGC) activator, BAY 41-8543, a derivative of riociguat, reduced the blood pressure to wild-type levels, suggesting that activation of sGC could be a treatment option for patients with HTNB. The mutant PDE3A enzymes show increased enzyme activity, aberrant phosphorylation and an increased interaction with the adaptor protein 14-3-3 q. Furthermore, a new mutation affecting the region encoding the catalytic domain of PDE3A was identified. All patients carrying this mutation suffer from brachydactyly type E but not all of them have hypertension. This mutant enzyme does not increase PDE3A activity, further confirming that only mutations affecting the regulatory region of the PDE3A gene increase the hydrolytic activity of the enzyme. Lastly, mutations located in the regulatory region of the PDE3A gene protect the heart against cardiac remodeling, hypertrophy and heart failure. In conclusion, these findings could lead to the identification of novel therapeutic strategies targeting PDE3A signaling compartments for the treatment of HTNB and essential hypertension, as well as heart failure.Hypertonie stellt den Hauptrisikofaktor für kardiovaskulär bedingte Todesfälle weltweit dar. Autosomal-dominante Hypertonie mit Brachydaktylie (HTNB) ist ein salz-resistentes Syndrom, das nach dem Mendelschen Erbgang vererbt wird. Es zeichnet sich durch fortschreitende Hypertonie, Brachydaktylie Typ E und Hyperplasie der Blutgefäße aus und ähnelt der essentiellen Hypertonie. Unbehandelt versterben Patienten noch vor Vollendung ihres 50. Lebensjahres an den Folgen eines Schlaganfalls. Diese Studie liefert aussagekräftige Belege, dass Mutationen in der 15 Basenpaar (bp) langen regulatorischen Region des Phosphodiesterase 3A (PDE3A)- Gens HTNB verursachen. Eine neue humane Mutation der 15 bp Region des PDE3A-Gens wurde identifiziert. Repräsentativ für diese Mutation wurde mit Hilfe von CRISPR/Cas9 ein Rattenmodell generiert, welches eine Deletion von 9 bp in eben dieser regulatorischen Region aufweist. Diese ist somit zu der humanen Deletion von 3 bp analog und rekapituliert HTNB. Das mutierte PDE3A-Gen fördert Mechanismen, die die Proliferation von glatten Gefäßmuskelzellen (VSMC) und den peripheren Gefäßwiderstand erhöhen und somit Hypertonie verursachen; die Herzfunktion erscheint dagegen normal. PDE3A hydrolysiert zyklisches Adenosinmonophosphat (cAMP) und wird durch zyklisches Guanosinmonophosphat (cGMP) inhibiert. Die Behandlung der mutierten Ratten mit dem löslichen Guanylyl- Zyklase (sGC)-Aktivator BAY 41-8543, einem Derivat von Riociguat, führte zu einer Reduktion des Blutdrucks auf Wildtyp-Level. Dies suggeriert, dass die Aktivierung von sGC eine Behandlungsoption für Patienten mit HTNB darstellen könnte. Die mutierten PDE3A-Enzyme zeigen erhöhte Enzymaktivität, aberrante Phosphorylierung und eine vermehrte Interaktion mit dem Adapterprotein 14-3-3 q. Des Weiteren wurde eine neue Mutation in der für die katalytische Domäne von PDE3A kodierenden Region identifiziert. Alle Träger dieser Mutation leiden unter Brachydaktylie Typ E, aber nicht alle weisen Hypertonie auf. Das mutierte Enzym erhöht nicht die PDE3A-Aktivität, was weiterhin bestätigt, dass nur die regulatorische Region des PDE3A-Gens betreffende Mutationen die hydrolytische Aktivität des Enzyms steigern. Zuletzt schützen Mutationen in der regulatorischen Region des PDE3A-Gens das Herz gegen Remodellierung, Hypertrophy und Herzversagen. Zusammenfassend könnten diese Erkenntnisse zur Identifizierung neuer therapeutischer Strategien führen, welche PDE3A-Signalkompartimente als Ziel haben und zur Behandlung von HTNB und essentieller Hypertonie, sowie Herzversagen eingesetzt werden könnten

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy

Background: Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Methods: Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. Results: We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. Conclusions: We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors

MYCN and HDAC5 transcriptionally repress CD9 to trigger invasion and metastasis in neuroblastoma

The systemic and resistant nature of metastatic neuroblastoma renders it largely incurable with current multimodal treatment. Clinical progression stems mainly from the increasing burden of metastatic colonization. Therapeutically inhibiting the migration-invasion-metastasis cascade would be of great benefit, but the mechanisms driving this cycle are as yet poorly understood. In-depth transcriptome analyses and ChIP-qPCR identified the cell surface glycoprotein, CD9, as a major downstream player and direct target of the recently described GRHL1 tumor suppressor. CD9 is known to block or facilitate cancer cell motility and metastasis dependent upon entity. High-level CD9 expression in primary neuroblastomas correlated with patient survival and established markers for favorable disease. Low-level CD9 expression was an independent risk factor for adverse outcome. MYCN and HDAC5 colocalized to the CD9 promoter and repressed transcription. CD9 expression diminished with progressive tumor development in the TH-MYCN transgenic mouse model for neuroblastoma, and CD9 expression in neuroblastic tumors was far below that in ganglia from wildtype mice. Primary neuroblastomas lacking MYCN amplifications displayed differential CD9 promoter methylation in methyl-CpG-binding domain sequencing analyses, and high-level methylation was associated with advanced stage disease, supporting epigenetic regulation. Inducing CD9 expression in a SH-EP cell model inhibited migration and invasion in Boyden chamber assays. Enforced CD9 expression in neuroblastoma cells transplanted onto chicken chorioallantoic membranes strongly reduced metastasis to embryonic bone marrow. Combined treatment of neuroblastoma cells with HDAC/DNA methyltransferase inhibitors synergistically induced CD9 expression despite hypoxic, metabolic or cytotoxic stress. Our results show CD9 is a critical and indirectly druggable suppressor of the invasion-metastasis cycle in neuroblastoma