MANCR drives esophageal carcinoma progression by targeting PDE4D.

PURPOSE: To explore the role of lncRNA MANCR in regulating in vitro proliferation and apoptosis in esophageal carcinoma cells and in vivo growth of esophageal carcinoma in nude mice. METHODS: MANCR levels in 15 pairs of esophageal carcinomas and non-tumoral tissues were detected by qRT-PCR. In vitro regulations of MANCR on proliferative and apoptotic potentials in TE-1 and EC-109 cells were explored by CCK-8, colony formation assay and flow cytometry. In addition, dual-luciferase reporter assay and rescue experiments were conducted to clarify the potential mechanisms of MANCR on regulating PDE4D. Finally, in vivo role of MANCR in mediating esophageal carcinoma growth was determined in nude mice implanted with EC-109 cells. RESULTS: MANCR was highly expressed in esophageal carcinomas tissues than non-tumoral ones. MANCR promoted proliferative ability and inhibited apoptosis in TE-1 and EC-109 cells. In nude mice with xenografted esophageal carcinoma, knockdown of MANCR markedly slowed down tumor growth. PDE4D was the target gene binding MANCR, which was downregulated in esophageal carcinoma tissues. Its level was negatively regulated by MANCR. Importantly, PDE4D could abolish the role of MANCR in stimulating the malignant progression of esophageal carcinoma. CONCLUSIONS: LncRNA MANCR is upregulated in esophageal carcinoma cases. Through negatively regulating PDE4D level, MANCR stimulates proliferative ability and inhibits apoptosis in esophageal carcinoma, thus driving the malignant progression.

The role of phosphodiesterase 4 in the pathophysiology of atopic dermatitis and the perspective for its inhibition.

Atopic dermatitis (AD) is a highly prevalent, chronic inflammatory skin disease that affects children and adults. The pathophysiology of AD is complex and involves skin barrier and immune dysfunction. Many immune cytokine pathways are amplified in AD, including T helper (Th) 2, Th22, Th17 and Th1. Current treatment guidelines recommend topical medications as initial therapy; however, until recently, only two drug classes were available: topical corticosteroids (TCSs) and topical calcineurin inhibitors (TCIs). Several limitations are associated with these agents. TCSs can cause a wide range of adverse effects, including skin atrophy, telangiectasia, rosacea and acne. TCIs can cause burning and stinging, and the prescribing information lists a boxed warning for a theoretical risk of malignancy. Novel medications with new mechanisms of action are necessary to provide better long-term control of AD. Phosphodiesterase 4 (PDE4) regulates cyclic adenosine monophosphate in cells and has been shown to be involved in the pathophysiology of AD, making it an attractive therapeutic target. Several PDE4 inhibitors are in clinical development for use in the treatment of AD, including crisaborole, which recently became the first topical PDE4 inhibitor approved for treatment of mild to moderate AD. This review will further describe the pathophysiology of AD, explain the possible role of PDE4 in AD and review PDE4 inhibitors currently approved or being investigated for use in AD.

Phosphodiesterase 4B plays a role in benzophenone-3-induced phototoxicity in normal human keratinocytes.

Benzophenone-3 (BP-3), which is extensively used in organic sunscreen, has phototoxic potential in human skin. Phosphodiesterase 4B (PDE4B) has a well-established role in inflammatory responses in immune cells. Currently, it is unknown if PDE4B is associated with BP-3-induced phototoxicity in normal human keratinocytes (NHKs). We found that BP-3 significantly increased PDE4B expression in ultraviolet B (UVB)-irradiated NHKs. Notably, BP-8, a sunscreen agent that shares the 2-hydroxy-4-methoxyphenyl methanone moiety with BP-3, also upregulated PDE4B expression in NHKs. Upon UVB irradiation, BP-3 upregulated the expression of pro-inflammatory factors, such as prostaglandin endoperoxide synthase 2, tumor necrosis factor α, interleukin 8, and S100A7, and downregulated the level of cornified envelope associated proteins, which are important in the development of the epidermal permeability barrier. The additive effects of UVB-activated BP-3 on the expression of both pro-inflammatory mediators and cornified envelope associated proteins were antagonized by treatment with the PDE4 inhibitor rolipram. The BP-3 and UVB co-stimulation-induced PDE4B upregulation and its association with the upregulation of pro-inflammatory mediators and the downregulation of epidermal differentiation markers were confirmed in a reconstituted three dimensional human epidermis model. Therefore, PDE4B has a role in the mechanism of BP-3-induced phototoxicity.

Transcription Factor Trps1 Promotes Tubular Cell Proliferation after Ischemia-Reperfusion Injury through cAMP-Specific 3',5'-Cyclic Phosphodiesterase 4D and AKT.

Trichorhinophalangeal 1 (Trps1) is a transcription factor essential for epithelial cell morphogenesis during kidney development, but the role of Trps1 in AKI induced by ischemia-reperfusion (I/R) remains unclear. Our study investigated Trps1 expression during kidney repair after acute I/R in rats and explored the molecular mechanisms by which Trps1 promotes renal tubular epithelial cell proliferation. Trps1 expression positively associated with the extent of renal repair after I/R injury. Compared with wild-type rats, rats with knockdown of Trps1 exhibited significantly delayed renal repair in the moderate I/R model, with lower GFR levels and more severe morphologic injury, whereas rats overexpressing Trps1 exhibited significantly accelerated renal repair after severe I/R injury. Additionally, knockdown of Trps1 inhibited and overexpression of Trps1 enhanced the proliferation of renal tubular epithelial cells in rats. Chromatin immunoprecipitation sequencing assays and RT-PCR revealed that Trps1 regulated cAMP-specific 3',5'-cyclic phosphodiesterase 4D (Pde4d) expression. Knockdown of Trps1 decreased the renal protein expression of Pde4d and phosphorylated Akt in rats, and dual luciferase analysis showed that Trps1 directly activated Pde4d transcription. Furthermore, knockdown of Pde4d or treatment with the phosphatidylinositol 3 kinase inhibitor wortmannin significantly inhibited Trps1-induced tubular cell proliferation in vitro Trps1 may promote tubular cell proliferation through the Pde4d/phosphatidylinositol 3 kinase/AKT signaling pathway, suggesting Trps1 as a potential therapeutic target for kidney repair after I/R injury.

Triple negative breast cancer development can be selectively suppressed by sustaining an elevated level of cellular cyclic AMP through simultaneously blocking its efflux and decomposition.

Triple negative breast cancer (TNBC) has the highest mortality among all breast cancer types and lack of targeted therapy is a key factor contributing to its high mortality rate. In this study, we show that 8-bromo-cAMP, a cyclic adenosine monophosphate (cAMP) analog at high concentration (> 1 mM) selectively suppresses TNBC cell growth. However, commonly-used cAMP-elevating agents such as adenylyl cyclase activator forskolin and pan phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) are ineffective. Inability of cAMP elevating agents to inhibit TNBC cell growth is due to rapid diminution of cellular cAMP through efflux and decomposition. By performing bioinformatics analyses with publically available gene expression datasets from breast cancer patients/established breast cancer cell lines and further validating using specific inhibitors/siRNAs, we reveal that multidrug resistance-associated protein 1/4 (MRP1/4) mediate rapid cAMP efflux while members PDE4 subfamily facilitate cAMP decomposition. When cAMP clearance is prevented by specific inhibitors, forskolin blocks TNBC's in vitro cell growth by arresting cell cycle at G1/S phase. Importantly, cocktail of forskolin, MRP inhibitor probenecid and PDE4 inhibitor rolipram suppresses TNBC in vivo tumor development. This study suggests that a TNBC-targeted therapeutic strategy can be developed by sustaining an elevated level of cAMP through simultaneously blocking its efflux and decomposition.

[Cyclic nucleotide phosphodiesterases: role in the heart and therapeutic perspectives]. / Phosphodiestérases des nucléotides cycliques : rôle dans le cœur et potentiel thérapeutique.

Cyclic nucleotide phosphodiesterases (PDEs) degrade the second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), thereby regulating multiple aspects of cardiac function. This highly diverse class of enzymes encoded by 21 genes encompasses 11 families that are not only responsible for the termination of cyclic nucleotide signalling, but are also involved in the generation of dynamic microdomains of cAMP and cGMP, controlling specific cell functions in response to various neurohormonal stimuli. In the myocardium, the PDE3 and PDE4 families predominate, degrading cAMP and thereby regulating cardiac excitation-contraction coupling. PDE3 inhibitors are positive inotropes and vasodilators in humans, but their use is limited to acute heart failure and intermittent claudication. PDE5 inhibitors, which are used with success to treat erectile dysfunction and pulmonary hypertension, do not seem efficient in heart failure with preserved ejection fraction. There is experimental evidence however that these PDE, as well as other PDE families including PDE1, PDE2 and PDE9, may play important roles in cardiac diseases, such as hypertrophy and heart failure (HF). After a brief presentation of the cyclic nucleotide pathways in cardiac myocytes and the major characteristics of the PDE superfamily, this review will focus on the potential use of PDE inhibitors in HF, and the recent research developments that could lead to a better exploitation of the therapeutic potential of these enzymes in the future.

Specific Inhibition of Phosphodiesterase-4B Results in Anxiolysis and Facilitates Memory Acquisition.

Cognitive dysfunction is a core feature of dementia and a prominent feature in psychiatric disease. As non-redundant regulators of intracellular cAMP gradients, phosphodiesterases (PDE) mediate fundamental aspects of brain function relevant to learning, memory, and higher cognitive functions. Phosphodiesterase-4B (PDE4B) is an important phosphodiesterase in the hippocampal formation, is a major Disrupted in Schizophrenia 1 (DISC1) binding partner and is itself a risk gene for psychiatric illness. To define the effects of specific inhibition of the PDE4B subtype, we generated mice with a catalytic domain mutant form of PDE4B (Y358C) that has decreased ability to hydrolyze cAMP. Structural modeling predictions of decreased function and impaired binding with DISC1 were confirmed in cell assays. Phenotypic characterization of the PDE4B(Y358C) mice revealed facilitated phosphorylation of CREB, decreased binding to DISC1, and upregulation of DISC1 and ß-Arrestin in hippocampus and amygdala. In behavioral assays, PDE4B(Y358C) mice displayed decreased anxiety and increased exploration, as well as cognitive enhancement across several tests of learning and memory, consistent with synaptic changes including enhanced long-term potentiation and impaired depotentiation ex vivo. PDE4B(Y358C) mice also demonstrated enhanced neurogenesis. Contextual fear memory, though intact at 24 h, was decreased at 7 days in PDE4B(Y358C) mice, an effect replicated pharmacologically with a non-selective PDE4 inhibitor, implicating cAMP signaling by PDE4B in a very late phase of consolidation. No effect of the PDE4B(Y358C) mutation was observed in the prepulse inhibition and forced swim tests. Our data establish specific inhibition of PDE4B as a promising therapeutic approach for disorders of cognition and anxiety, and a putative target for pathological fear memory.

Ferulic acid alleviates Aß25-35- and lipopolysaccharide-induced PC12 cellular damage: a potential role in Alzheimer's disease by PDE inhibition.

OBJECTIVE: Phosphodiesterase (PDE) plays an important role in the pathogenesis of Alzheimer's disease (AD). Ferulic acid (FA) has a therapeutic benefit in the treatment of AD. We investigated whether this therapeutic effect is based on the modulation of the PDE/cyclic adenosine monophosphate (cAMP) pathway. In the present study, we investigated whether FA could abrogate Aß25-35- and lipopolysaccharide-induced cellular damage. MATERIALS AND METHODS: Cell viability, superoxide production, and the levels of inflammatory factors were investigated. We further investigated the intracellular levels of cAMP and Ca2+, both of which are associated with PDE activity. Furthermore, molecular docking was used to identify the binding mode between phosphodiesterase 4B2 (PDE4B2) and FA. RESULTS: Pretreatment with FA significantly maintained cell viability, increased the levels of superoxide dismutase, and inhibited production of TNF-α and IL-1ß induced by Aß25-35. Moreover, pretreatment with FA increased the intracellular levels of cAMP and decreased the intracellular levels of Ca2+. The docking results also showed that FA has the potential to inhibit PDE4B2 activity. CONCLUSIONS: Taken together, our results suggested that one of the therapeutic effects of FA on AD was potentially mediated by modulating the PDE/cAMP pathway.

Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4.

BACKGROUND AND PURPOSE: This study examined the role of the main vascular cAMP-hydrolysing phosphodiesterases (cAMP-PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by ß-adrenoceptors, following heart failure (HF). EXPERIMENTAL APPROACH: Twenty-two weeks after proximal aortic stenosis, to induce HF, or SHAM surgery in rats, we evaluated the expression, activity and function of cAMP-PDE in the descending thoracic aorta. KEY RESULTS: HF rat aortas exhibited signs of endothelial dysfunction, with alterations of the NO pathway, and alteration of PDE3 and PDE4 subtype expression, without changing total aortic cAMP-hydrolytic activity and PDE1, PDE3 and PDE4 activities. Vascular reactivity experiments using PDE inhibitors showed that PDE3 and PDE4 controlled the level of PGF2α -stimulated contraction in SHAM aorta. PDE3 function was partially inhibited by endothelial NO, whereas PDE4 function required a functional endothelium and was under the negative control of PDE3. In HF, PDE3 function was preserved, but its regulation by endothelial NO was altered. PDE4 function was abolished and restored by PDE3 inhibition. In PGF2α -precontracted arteries, ß-adrenoceptor stimulation-induced relaxation in SHAM aorta, which was abolished in the absence of functional endothelium, as well as in HF aortas, but restored after PDE3 inhibition in all unresponsive arteries. CONCLUSIONS AND IMPLICATIONS: Our study underlines the key role of the endothelium in controlling the contribution of smooth muscle PDE to contractile function. In HF, endothelial dysfunction had a major effect on PDE3 function and PDE3 inhibition restored a functional relaxation to ß-adrenoceptor stimulation.

Prostaglandin E2 does not attenuate adrenergic-induced cardiac contractile response.

Systemic inflammation may contribute to heart failure. PGE2 was recently suggested to mediate inflammation-induced impairment of cardiac function by desensitizing the murine heart to isoprenaline. Given the magnitude of the reported effect and the potential relevance, we sought to reproduce it in the human heart. Human trabeculae were prepared from the right atrial tissue obtained during heart surgery and from the right ventricle of two explanted human failing hearts. Muscle strips were electrically driven and isometric force development was measured. PGE2 was given at a single concentration (0.1 µM). Norepinephrine was used to activate ß1-adrenoceptors, epinephrine to activate ß2-adrenoceptors in atrial trabeculae. Isoprenaline was used in ventricular tissue. All patients were in sinus rhythm. Murine ventricular strips were used for comparison and stimulated with isoprenaline. The pharmacological activity of the PGE2 batch was confirmed by assessing concentration-dependent vasoconstriction in murine aorta. We used atrial and ventricular trabeculae from humans. Exposure to PGE2 (15 min) did not affect contractility when compared to time-matched controls. PGE2 neither altered the sensitivity or efficacy of ß1- or ß2-adrenoceptor-mediated stimulation of force in human atrial or in ventricular trabeculae for nonselective ß1- or ß2-adrenoceptor-stimulation. Surprisingly, PGE2 also did not affect -logEC50 values or maximum catecholamine-stimulated force in ventricular strips from mice, whereas it induced vasoconstriction in aortic rings with an -logEC50 of 5.0 (n = 6). Our data do not support a role for PGE2 in regulating catecholamine inotropy, neither in mice nor in humans.

Carvedilol induces greater control of ß2- than ß 1-adrenoceptor-mediated inotropic and lusitropic effects by PDE3, while PDE4 has no effect in human failing myocardium.

The ß-blockers carvedilol and metoprolol provide important therapeutic strategies for heart failure treatment. Therapy with metoprolol facilitates the control by phosphodiesterase PDE3, but not PDE4, of inotropic effects of catecholamines in human failing ventricle. However, it is not known whether carvedilol has the same effect. We investigated whether the PDE3-selective inhibitor cilostamide (0.3 µM) or PDE4-selective inhibitor rolipram (1 µM) modified the positive inotropic and lusitropic effects of catecholamines in ventricular myocardium of heart failure patients treated with carvedilol. Right ventricular trabeculae from explanted hearts of nine carvedilol-treated patients with terminal heart failure were paced to contract at 1 Hz. The effects of (-)-noradrenaline, mediated through ß1-adrenoceptors (ß2-adrenoceptors blocked with ICI118551), and (-)-adrenaline, mediated through ß2-adrenoceptors (ß1-adrenoceptors blocked with CGP20712A), were assessed in the absence and presence of the PDE inhibitors. The inotropic potency, estimated from -logEC50s, was unchanged for (-)-noradrenaline but decreased 16-fold for (-)-adrenaline in carvedilol-treated compared to non-ß-blocker-treated patients, consistent with the previously reported ß2-adrenoceptor-selectivity of carvedilol. Cilostamide caused 2- to 3-fold and 10- to 35-fold potentiations of the inotropic and lusitropic effects of (-)-noradrenaline and (-)-adrenaline, respectively, in trabeculae from carvedilol-treated patients. Rolipram did not affect the inotropic and lusitropic potencies of (-)-noradrenaline or (-)-adrenaline. Treatment of heart failure patients with carvedilol induces PDE3 to selectively control the positive inotropic and lusitropic effects mediated through ventricular ß2-adrenoceptors compared to ß1-adrenoceptors. The ß2-adrenoceptor-selectivity of carvedilol may provide protection against ß2-adrenoceptor-mediated ventricular overstimulation in PDE3 inhibitor-treated patients. PDE4 does not control ß1- and ß2-adrenoceptor-mediated inotropic and lusitropic effects in carvedilol-treated patients.

Mice deficient in phosphodiesterase-4A display anxiogenic-like behavior.

RATIONALE: Phosphodiesterases (PDEs) are a super family of enzymes responsible for the halting of intracellular cyclic nucleotide signaling and may represent novel therapeutic targets for treatment of cognitive disorders. PDE4 is of considerable interest to cognitive research because it is highly expressed in the brain, particularly in the cognition-related brain regions. Recently, the functional role of PDE4B and PDE4D, two of the four PDE4 subtypes (PDE4A, B, C, and D), in behavior has begun to be identified; however, the role of PDE4A in the regulation of behavior is still unknown. OBJECTIVES: The purpose of this study was to characterize the functional role of PDE4A in behavior. METHODS: The role of PDE4A in behavior was evaluated through a battery of behavioral tests using PDE4A knockout (KO) mice; urine corticosterone levels were also measured. RESULTS: PDE4A KO mice exhibited improved memory in the step-through-passive-avoidance test. They also displayed anxiogenic-like behavior in elevated-plus maze, holeboard, light-dark transition, and novelty suppressed feeding tests. Consistent with the anxiety profile, PDE4A KO mice had elevated corticosterone levels compared with wild-type controls post-stress. Interestingly, PDE4A KO mice displayed no change in object recognition, Morris water maze, forced swim, tail suspension, and duration of anesthesia induced by co-administration of xylazine and ketamine (suggesting that PDE4A KO may not be emetic). CONCLUSIONS: These results suggest that PDE4A may be important in the regulation of emotional memory and anxiety-like behavior, but not emesis. PDE4A could possibly represent a novel therapeutic target in the future for anxiety or disorders affecting memory.

PDE4B mediates local feedback regulation of ß1-adrenergic cAMP signaling in a sarcolemmal compartment of cardiac myocytes.

Multiple cAMP phosphodiesterase (PDE) isoforms play divergent roles in cardiac homeostasis but the molecular basis for their non-redundant function remains poorly understood. Here, we report a novel role for the PDE4B isoform in ß-adrenergic (ßAR) signaling in the heart. Genetic ablation of PDE4B disrupted ßAR-induced cAMP transients, as measured by FRET sensors, at the sarcolemma but not in the bulk cytosol of cardiomyocytes. This effect was further restricted to a subsarcolemmal compartment because PDE4B regulates ß1AR-, but not ß2AR- or PGE2-induced responses. The spatially restricted function of PDE4B was confirmed by its selective effects on PKA-mediated phosphorylation patterns. PDE4B limited the PKA-mediated phosphorylation of key players in excitation-contraction coupling that reside in the sarcolemmal compartment, including L-type Ca(2+) channels and ryanodine receptors, but not phosphorylation of distal cytosolic proteins. ß1AR- but not ß2AR-ligation induced PKA-dependent activation of PDE4B and interruption of this negative feedback with PKA inhibitors increased sarcolemmal cAMP. Thus, PDE4B mediates a crucial PKA-dependent feedback that controls ß1AR-dependent cAMP signals in a restricted subsarcolemmal domain. Disruption of this feedback augments local cAMP/PKA signals, leading to an increased intracellular Ca(2+) level and contraction rate.

Differential regulation of cardiac excitation-contraction coupling by cAMP phosphodiesterase subtypes.

AIMS: Multiple phosphodiesterases (PDEs) hydrolyze cAMP in cardiomyocytes, but the functional significance of this diversity is not well understood. Our goal here was to characterize the involvement of three different PDEs (PDE2-4) in cardiac excitation-contraction coupling (ECC). METHODS AND RESULTS: Sarcomere shortening and Ca(2+) transients were recorded simultaneously in adult rat ventricular myocytes and ECC protein phosphorylation by PKA was determined by western blot analysis. Under basal conditions, selective inhibition of PDE2 or PDE3 induced a small but significant increase in Ca(2+) transients, sarcomere shortening, and troponin I phosphorylation, whereas PDE4 inhibition had no effect. PDE3 inhibition, but not PDE2 or PDE4, increased phospholamban phosphorylation. Inhibition of either PDE2, 3, or 4 increased phosphorylation of the myosin-binding protein C, but neither had an effect on L-type Ca(2+) channel or ryanodine receptor phosphorylation. Dual inhibition of PDE2 and PDE3 or PDE2 and PDE4 further increased ECC compared with individual PDE inhibition, but the most potent combination was obtained when inhibiting simultaneously PDE3 and PDE4. This combination also induced a synergistic induction of ECC protein phosphorylation. Submaximal ß-adrenergic receptor stimulation increased ECC, and this effect was potentiated by individual PDE inhibition with the rank order of potency PDE4 = PDE3 > PDE2. Identical results were obtained on ECC protein phosphorylation. CONCLUSION: Our results demonstrate that PDE2, PDE3, and PDE4 differentially regulate ECC in adult cardiomyocytes. PDE2 and PDE3 play a more prominent role than PDE4 in regulating basal cardiac contraction and Ca(2+) transients. However, PDE4 becomes determinant when cAMP levels are elevated, for instance, upon ß-adrenergic stimulation or PDE3 inhibition.

Rolipram attenuates bile duct ligation-induced liver injury in rats: a potential pathogenic role of PDE4.

Anti-inflammatory and antifibrotic effects of the broad spectrum phosphodiesterase (PDE) inhibitor pentoxifylline have suggested an important role for cyclic nucleotides in the pathogenesis of hepatic fibrosis; however, studies examining the role of specific PDEs are lacking. Endotoxemia and Toll-like receptor 4 (TLR4)-mediated inflammatory and profibrotic signaling play a major role in the development of hepatic fibrosis. Because cAMP-specific PDE4 critically regulates lipopolysaccharide (LPS)-TLR4-induced inflammatory cytokine expression, its pathogenic role in bile duct ligation-induced hepatic injury and fibrogenesis in Sprague-Dawley rats was examined. Initiation of cholestatic liver injury and fibrosis was accompanied by a significant induction of PDE4A, B, and D expression and activity. Treatment with the PDE4-specific inhibitor rolipram significantly decreased liver PDE4 activity, hepatic inflammatory and profibrotic cytokine expression, injury, and fibrosis. At the cellular level, in relevance to endotoxemia and inflammatory cytokine production, PDE4B was observed to play a major regulatory role in the LPS-inducible tumor necrosis factor (TNF) production by isolated Kupffer cells. Moreover, PDE4 expression was also involved in the in vitro activation and transdifferentiation of isolated hepatic stellate cells (HSCs). Particularly, PDE4A, B, and D upregulation preceded induction of the HSC activation marker α-smooth muscle actin (α-SMA). In vitro treatment of HSCs with rolipram effectively attenuated α-SMA, collagen expression, and accompanying morphologic changes. Overall, these data strongly suggest that upregulation of PDE4 expression during cholestatic liver injury plays a potential pathogenic role in the development of inflammation, injury, and fibrosis.

[Role of cyclic nucleotide phosphodiesterases type 3 and 4 in cardiac excitation-contraction coupling and arrhythmias]. / Rôle des phosphodiestérases des nucléotides cycliques de types 3 et 4 dans le couplage excitation-contraction et les arythmies cardiaques.

Cyclic nucleotide phosphodiesterases (PDE) represent a superfamily of enzymes specialised in the degradation of cAMP and cGMP. In heart, PDE3 and PDE4 are the two major families involved in the regulation of cAMP levels and the control of inotropism. Both families are encoded by several genes, and the recent analysis of the cardiac phenotype of mice lacking these different genes provided new insights into the way they regulate excitation-contraction coupling (ECC). In particular, these studies emphasize the local character of ECC regulation by PDE, as well as the role of these PDE in maintaining calcium homeostasis and preventing cardiac arrhythmias.

Influence of phosphodiesterases and cGMP on cAMP generation and on phosphorylation of phospholamban and troponin I by 5-HT4 receptor activation in porcine left atrium.

Our objective was to investigate the role of phosphodiesterase (PDE)3 and PDE4 and cGMP in the control of cAMP metabolism and of phosphorylation of troponin I (TnI) and phospholamban (PLB) when 5-HT4 receptors are activated in pig left atrium. Electrically paced porcine left atrial muscles, mounted in organ baths, received stimulators of particulate guanylyl cyclase (pGC) or soluble guanylyl cyclase (sGC) and/or specific PDE inhibitors followed by 5-HT or the 5-HT4 receptor agonist prucalopride. Muscles were freeze-clamped at different moments of exposure to measure phosphorylation of the cAMP/protein kinase A targets TnI and PLB by immunoblotting and cAMP levels by enzyme immunoassay. Corresponding with the functional results, 5-HT only transiently increased cAMP content, but caused a less quickly declining phosphorylation of PLB and did not significantly change TnI phosphorylation. Under combined PDE3 and PDE4 inhibition, the 5-HT-induced increase in cAMP levels and PLB phosphorylation was enhanced and sustained, and TnI phosphorylation was now also increased. Responses to prucalopride per se and the influence thereupon of PDE3 and PDE4 inhibition were similar except that responses were generally smaller. Stimulation of pGC together with PDE4 inhibition increased 5-HT-induced PLB phosphorylation compared to 5-HT alone, consistent with functional responses. sGC stimulation hastened the fade of inotropic responses to 5-HT, while cAMP levels were not altered. PDE3 and PDE4 control the cAMP response to 5-HT4 receptor activation, causing a dampening of downstream signalling. Stimulation of pGC is able to enhance inotropic responses to 5-HT by increasing cAMP levels, while sGC stimulation decreases contraction to 5-HT cAMP independently.

Phosphodiesterase-4 promotes proliferation and angiogenesis of lung cancer by crosstalk with HIF.

Lung cancer is the leading cause of cancer death worldwide. Recent data suggest that cyclic nucleotide phosphodiesterases (PDEs) are relevant in various cancer pathologies. Pathophysiological role of phosphodiesterase 4 (PDE4) with possible therapeutic prospects in lung cancer was investigated. We exposed 10 different lung cancer cell lines (adenocarcinoma, squamous and large cell carcinoma) to hypoxia and assessed expression and activity of PDE4 by real-time PCR, immunocytochemistry, western blotting and PDE activity assays. Expression and activity of distinct PDE4 isoforms (PDE4A and PDE4D) increased in response to hypoxia in eight of the studied cell lines. Furthermore, we analyzed various in silico predicted hypoxia-responsive elements (p-HREs) found in in PDE4A and PDE4D genes. Performing mutation analysis of the p-HRE in luciferase reporter constructs, we identified four functional HRE sites in the PDE4A gene and two functional HRE sites in the PDE4D gene that mediated hypoxic induction of the reporter. Silencing of hypoxia-inducible factor subunits (HIF1α and HIF2α) by small interfering RNA reduced hypoxic induction of PDE4A and PDE4D. Vice versa, using a PDE4 inhibitor (PDE4i) as a cyclic adenosine monophosphate (cAMP) -elevating agent, cAMP analogs or protein kinase A (PKA)-modulating drugs and an exchange protein directly activated by cAMP (EPAC) activator, we demonstrated that PDE4-cAMP-PKA/EPAC axis enhanced HIF signaling as measured by HRE reporter gene assay, HIF and HIF target genes expression ((lactate dehydrogenase A), LDHA, (pyruvate dehydrogenase kinase 1) PDK1 and (vascular endothelial growth factor A) VEGFA). Notably, inhibition of PDE4 by PDE4i or silencing of PDE4A and PDE4D reduced human lung tumor cell proliferation and colony formation. On the other hand, overexpression of PDE4A or PDE4D increased human lung cancer proliferation. Moreover, PDE4i treatment reduced hypoxia-induced VEGF secretion in human cells. In vivo, PDE4i inhibited tumor xenograft growth in nude mice by attenuating proliferation and angiogenesis. Our findings suggest that PDE4 is expressed in lung cancer, crosstalks with HIF signaling and promotes lung cancer progression. Thus, PDE4 may represent a therapeutic target for lung cancer therapy.

Effect of oxygen on phosphodiesterases (PDE) 3 and 4 isoforms and PKA activity in the superior cervical ganglia.

UNLABELLED: The cAMP-protein kinase A (PKA) signaling pathway is involved in regulating the release of transmitters from neurons and other cells. Multiple phosphodiesterase (PDE) isoforms regulate this pathway, however, the pattern of isoform expression and stimulus response across tissues has not been fully characterized.Using fluorescent resonance energy transfer (FRET)-based imaging in primary superior cervical ganglia (SCG) neurons and real-time qPCR, we explored the role of PDE3 and PDE4 isoforms and oxygen tension in the activation of PKA and changes in gene expression. These primary neurons were infected with an adenovirus containing A-Kinase activity reporter (AKAR3) and assayed for responses to PDE inhibitors: rolipram (ROL, 1 µM), milrinone (MIL, 10 µM) and IBMX (100 µM), and adenylyl cyclase activator forskolin (FSK, 50 µM). Different PDE activity patterns were observed in different cells: high PDE4 activity (n = 3), high PDE3 activity (n = 3) and presence of activity of other PDEs (n = 3). Addition of PKA inhibitor H89 (10 µM) completely reversed the response. We further studied the effect of oxygen in the PKA activity induced by PDE inhibition. Both normoxia (20%O(2)/5%CO(2)) and hypoxia (0%O(2)/5%CO(2)) induced a similar increase in the FRET emission ratio (14.5 ± 0.8 and 14.7 ± 0.8, respectively).PDE3a, PDE4b and PDE4d isoforms mRNAs were highly expressed in the whole SCG with no modulation by hypoxia. CONCLUSION: Using a FRET-based PKA activity sensor, we show that primary SCG neurons can be used as a model system to dissect the contribution of different PDE isoforms in regulating cAMP/PKA signaling. The differential patterns of PDE regulation potentially represent subpopulations of ganglion cells with different physiological functions.