Phosphodiesterase 8A Research Articles

Regulation of intracellular cAMP levels in osteocytes by mechano-sensitive focal adhesion kinase via PDE8A.

Osteocytes are the primary mechano-sensitive cell type in bone. Mechanical loading is sensed across the dendritic projections of osteocytes leading to transient reductions in focal adhesion kinase (FAK) activity. Knowledge regarding the signaling pathways downstream of FAK in osteocytes is incomplete. We performed tyrosine-focused phospho-proteomic profiling in osteocyte-like Ocy454 cells to identify FAK substrates. Gsα, parathyroid hormone receptor (PTH1R), and phosphodiesterase 8A (PDE8A), all proteins associated with cAMP signaling, were found as potential FAK targets based on their reduced tyrosine phosphorylation in both FAK- deficient or FAK inhibitor treated cells. Real time monitoring of intracellular cAMP levels revealed that FAK pharmacologic inhibition or gene deletion increased basal and GPCR ligand-stimulated cAMP levels and downstream phosphorylation of protein kinase A substrates. Mutating FAK phospho-acceptor sites in Gsα and PTH1R had no effect on PTH- or FAK inhibitor-stimulated cAMP levels. Since FAK inhibitor treatment augmented cAMP levels even in the presence of forskolin, we focused on potential FAK substrates downstream of cAMP generation. Indeed, PDE8A inhibition mimicked FAK inhibition at the level of increased cAMP, PKA activity, and expression of cAMP-regulated target genes. In vitro kinase assay showed that PDE8A is directly phosphorylated by FAK while immunoprecipitation assays revealed intracellular association between FAK and PDE8A. Thus, FAK inhibition in osteocytes acts synergistically with signals that activate adenylate cyclase to increase intracellular cAMP. Mechanically-regulated FAK can modulate intracellular cAMP levels via effects on PDE8A. These data suggest a novel signal transduction mechanism that mediates crosstalk between mechanical and cAMP-linked hormonal signaling in osteocytes.

14-3-3 interaction with phosphodiesterase 8A sustains PKA signaling and downregulates the MAPK pathway

The cAMP/PKA and mitogen-activated protein kinase (MAPK) signaling cascade control many cellular processes and are highly regulated for optimal cellular responses upon external stimuli. Phosphodiesterase 8A (PDE8A) is an important regulator that inhibits signaling via cAMP-dependent PKA by hydrolyzing intracellular cAMP pool. Conversely, PDE8A activates the MAPK pathway by protecting CRAF/Raf1 kinase from PKA-mediated inhibitory phosphorylation at Ser259 residue, a binding site of scaffold protein 14-3-3. It still remains enigmatic as to how the cross-talk involving PDE8A regulation influences cAMP/PKA and MAPK signaling pathways. Here, we report that PDE8A interacts with 14-3-3ζ in both yeast and mammalian system, and this interaction is enhanced upon the activation of PKA, which phosphorylates PDE8A's Ser359 residue. Biophysical characterization of phospho-Ser359 peptide with 14-3-3ζ protein further supports their interaction. Strikingly, 14-3-3ζ reduces the catalytic activity of PDE8A, which upregulates the cAMP/PKA pathway while the MAPK pathway is downregulated. Moreover, 14-3-3ζ in complex with PDE8A and cAMP-bound regulatory subunit of PKA, RIα, delays the deactivation of PKA signaling. Our results define 14-3-3ζ as a molecular switch that operates signaling between cAMP/PKA and MAPK by associating with PDE8A.

Uncovering the selectivity mechanism of phosphodiesterase 7A/8A inhibitors through computational studies.

The expression of phosphodiesterase 7A (PDE7A) and phosphodiesterase 8A (PDE8) genes is integral to human signaling pathways, and the inhibition of PDE7A has been associated with the onset of various diseases, including effects on the immune system and nervous system. The development of PDE7 selective inhibitors can promote research on immune and nervous system diseases, such as multiple sclerosis, chronic inflammation, and autoimmune responses. PDE8A is expressed alongside PDE8B, and its inhibitory mechanism is still unclear. Studying the mechanisms of selective inhibitors against different PDE subtypes is crucial to prevent potential side effects, such as nausea and cardiac toxicity, and the sequence similarity of the two protein subtypes was 55.9%. Therefore, it is necessary to investigate the differences of both subtypes' ligand binding sites. Selective inhibitors of two proteins were chosen to summarize the reason for their selectivity through molecular docking, molecular dynamics simulation, alanine scanning mutagenesis, and MM-GBSA calculation. We found that Phe384PDE7A, Leu401PDE7A, Gln413PDE7A, Tyr419PDE7A, and Phe416PDE7A in the active site positively contribute to the selectivity towards PDE7A. Additionally, Asn729PDE8A, Phe767PDE8A, Gln778PDE8A, and Phe781PDE8A positively contribute to the selectivity towards PDE8A.

CAMP-specific phosphodiesterase 8A and 8B isoforms are differentially expressed in human testis and Leydig cell tumor.

Cyclic adenosine monophosphate/Protein kinase A (cAMP/PKA) signaling pathway is the master regulator of endocrine tissue function. The level, compartmentalization and amplitude of cAMP response are finely regulated by phosphodiesterases (PDEs). PDE8 is responsible of cAMP hydrolysis and its expression has been characterized in all steroidogenic cell types in rodents including adrenal and Leydig cells in rodents however scarce data are currently available in humans. Here we demonstrate that human Leydig cells express both PDE8A and PDE8B isoforms. Interestingly, we found that the expression of PDE8B but not of PDE8A is increased in transformed Leydig cells (Leydig cell tumors-LCTs) compared to non-tumoral cells. Immunofluorescence analyses further reveals that PDE8A is also highly expressed in specific spermatogenic stages. While the protein is not detected in spermatogonia it accumulates nearby the forming acrosome, in the trans-Golgi apparatus of spermatocytes and spermatids and it follows the fate of this organelle in the later stages translocating to the caudal part of the cell. Taken together our findings suggest that 1) a specific pool(s) of cAMP is/are regulated by PDE8A during spermiogenesis pointing out a possible new role of this PDE8 isoform in key events governing the differentiation and maturation of human sperm and 2) PDE8B can be involved in Leydig cell transformation.

Phosphodiesterase 8A to discriminate in blood samples depressed patients and suicide attempters from healthy controls based on A-to-I RNA editing modifications

Mental health issues, including major depressive disorder, which can lead to suicidal behavior, are considered by the World Health Organization as a major threat to global health. Alterations in neurotransmitter signaling, e.g., serotonin and glutamate, or inflammatory response have been linked to both MDD and suicide. Phosphodiesterase 8A (PDE8A) gene expression is significantly decreased in the temporal cortex of major depressive disorder (MDD) patients. PDE8A specifically hydrolyzes adenosine 3′,5′-cyclic monophosphate (cAMP), which is a key second messenger involved in inflammation, cognition, and chronic antidepressant treatment. Moreover, alterations of RNA editing in PDE8A mRNA has been described in the brain of depressed suicide decedents. Here, we investigated PDE8A A-to-I RNA editing-related modifications in whole blood of depressed patients and suicide attempters compared to age-matched and sex-matched healthy controls. We report significant alterations of RNA editing of PDE8A in the blood of depressed patients and suicide attempters with major depression, for which the suicide attempt took place during the last month before sample collection. The reported RNA editing modifications in whole blood were similar to the changes observed in the brain of suicide decedents. Furthermore, analysis and combinations of different edited isoforms allowed us to discriminate between suicide attempters and control groups. Altogether, our results identify PDE8A as an immune response-related marker whose RNA editing modifications translate from brain to blood, suggesting that monitoring RNA editing in PDE8A in blood samples could help to evaluate depressive state and suicide risk.

Rational Design of 2-Chloroadenine Derivatives as Highly Selective Phosphodiesterase 8A Inhibitors.

To validate the hypothesis that Tyr748 is a crucial residue to aid the discovery of highly selective phosphodiesterase 8A (PDE8A) inhibitors, we identified a series of 2-chloroadenine derivatives based on the hit clofarabine. Structure-based design targeting Tyr748 in PDE8 resulted in the lead compound 3a (IC50 = 0.010 μM) with high selectivity with a reasonable druglike profile. In the X-ray crystal structure, 3a bound to PDE8A with a different mode from 3-isobutyl-1-methylxanthine (a pan-PDE inhibitor) and gave a H-bond of 2.7 Å with Tyr748, which possibly interprets the 220-fold selectivity of 3a against PDE2A. Additionally, oral administration of compound 3a achieved remarkable therapeutic effects against vascular dementia (VaD), indicating that PDE8 inhibitors could serve as potential anti-VaD agents.

Mitochondrial sub-cellular localization of cAMP-specific phosphodiesterase 8A in ovarian follicular cells

Cyclic adenosine monophosphate (cAMP) is a ubiquitous secondary messenger that plays a central role in endocrine tissue function, particularly in the synthesis of steroid hormones. The intracellular concentration of cAMP is regulated through its synthesis by cyclases and its degradation by cyclic nucleotide phosphodiesterases (PDEs). Although the expression and activity of PDEs impact the specificity and the amplitude of the cAMP response, it is becoming increasingly clear that the sub-cellular localization of PDE emphasizes the spatial regulation of the cell signalling processes that are essential for normal cellular function. We first examined the expression of PDE8A in porcine ovarian cells. PDE8A is expressed in granulosa cells, cumulus cells and oocytes. Second, we assessed the mitochondrial sub-cellular localization of PDE8A. Using western blotting with isolated mitochondrial fractions from granulosa cells and cumulus-oocyte complexes revealed immuno-reactive bands. PDE assay of isolated mitochondrial fractions from granulosa cells measured specific PDE8 cAMP-PDE activity as PF-04957325-sensitive. The immune-reactive PDE8A signal and MitoTracker labelling co-localized supporting mitochondrial sub-cellular localization of PDE8A, which was confirmed using immuno-electron microscopy. Finally, the effect of PDE8 on progesterone production was assessed during the in-vitro maturation of cumulus-oocyte complexes. Using PF-04957325, we observed a significant increase (P < 0.05) in progesterone secretion with follicle-stimulating hormone (FSH). Active mitochondria stained with MitoTracker orange CMTMRos were also increased by the specific PDE8 inhibitor supporting its functional regulation. In conclusion, we propose the occurrence of mitochondrial sub-cellular localization of PDE8A in porcine granulosa cells and cumulus cells. This suggests that there is potential for new strategies for ovarian stimulation and artificial reproductive technologies, as well as the possibility for using new media to improve the quality of oocytes.

RNA editing blood biomarkers for predicting mood alterations in HCV patients

Treatment-emergent depression is a common complication in patients with chronic hepatitis C virus (HCV) infection undergoing antiviral combination therapy with IFN-α and ribavirin. It has recently been shown that changes in A-to-I RNA editing rates are associated with various pathologies such as inflammatory disorders, depression and suicide. Interestingly, IFN-α induces gene expression of the RNA editing enzyme ADAR1-1 (ADAR1a-p150) and alters overall RNA editing activity. In this study, we took advantage of the high prevalence of pharmacologically induced depression in patients treated with IFN-α and ribavirin to test the interest of RNA editing–related biomarkers in white blood cells of patients. In this 16-week longitudinal study, a small cohort of patients was clinically evaluated using standard assessment methods prior to and during antiviral therapy and blood samples were collected to analyse RNA editing modifications. A-I RNA editing activity on the phosphodiesterase 8A (PDE8A) gene, a previously identified RNA editing hotspot in the context of lupus erythematosus, was quantified by using an ultra-deep next-generation sequencing approach. We also monitored gene expression levels of the ADAR enzymes and the PDE8A gene during treatment by qPCR. As expected, psychiatric evaluation could track treatment-emergent depression, which occurred in 30% of HCV patients. We show that PDE8A RNA editing is increased in all patients following interferon treatment, but differently in 30% of patients. This effect was mimicked in a cellular model using SHSY-5Y neuroblastoma cells. By combining the data of A-I RNA editing and gene expression, we generated an algorithm that allowed discrimination between the group of patients who developed a treatment-emergent depression and those who did not. The current model of drug-induced depression identified A-I RNA editing biomarkers as useful tools for the identification of individuals at risk of developing depression in an objective, quantifiable biological blood test.

Brain region-specific alterations of RNA editing in PDE8A mRNA in suicide decedents

Phosphodiesterases (PDE) are key modulators of signal transduction and are involved in inflammatory cell activation, memory and cognition. There is a two-fold decrease in the expression of phosphodiesterase 8A (PDE8A) in the temporal cortex of major depressive disorder (MDD) patients. Here, we studied PDE8A mRNA-editing profile in two architectonically distinct neocortical regions in a clinically well-characterized cohort of age- and sex-matched non-psychiatric drug-free controls and depressed suicide decedents. By using capillary electrophoresis single-stranded conformational polymorphism (CE-SSCP), a previously validated technique to identify A-to-I RNA modifications, we report the full editing profile of PDE8A in the brain, including identification of two novel editing sites. Editing of PDE8A mRNA displayed clear regional difference when comparing dorsolateral prefrontal cortex (BA9) and anterior cingulate cortex (BA24). Furthermore, we report significant intra-regional differences between non-psychiatric control individuals and depressed suicide decedents, which could discriminate the two populations. Taken together, our results (i) highlight the importance of immune/inflammatory markers in major depressive disorder and suicide and (ii) establish a direct relationship between A-to-I RNA modifications of peripheral markers and A-to-I RNA editing-related modifications in brain. This work provides the first immune response-related brain marker for suicide and could pave the way for the identification of a blood-based biomarker that predicts suicidal behavior.

P058 Phosphodiesterase 8A domains at the site of leucocyte–endothelial cell adhesion in ulcerative colitis submucosa

Development of anti-adhesion therapies like Etrolizumab and Alicaforsen highlights the potential for inhibiting recruitment pathways in ulcerative colitis (UC). Recent in vitro mouse work from our group shows that disruption of Phosphodiesterase (PDE) 8A domains inhibits T cell adhesion to ICAM-1 and endothelial cells.1 PDE inhibitors are known immunosuppressive agents, yet no PDE inhibitors are approved in UC due to dose-limiting side effects. As PDE8A is the most efficient in vivo hydrolyser of cAMP, PDE8A inhibitors are good candidates for overcoming the challenges presented mainly by PDE4 inhibitors. PDE8A expression in colorectal cancer (CRC) is well documented and PDE8A is reported in UC intestinal crypts by gene and protein array. However, the in situ localisation of PDE8A in UC submucosa during recruitment of leukocytes is unknown. Therefore, we developed an image analysis method to highlight PDE8A domains and investigated whether PDE8A domains were present at the site of leucocyte-endothelial cell adhesion in the submucosa of inflamed UC resections. PDE8A protein expression in five involved and two uninvolved UC colon resections was determined by immunohistochemistry using anti-PDE8A (ab61815) or IgG isotype (ab125938). Six paired biopsies from CRC (involved and uninvolved) were used as controls. Antibodies were titrated to 1:250 to reveal subcellular domains masked by diffuse staining at 1:100. Images were analysed using the saturation feature (0-255) in Image J software to assign PDE8A staining intensity as a representation of subcellular domains (Figure A). This in situ study performed in human UC tissue reveals PDE8A domains in the plasma membrane of submucosal leucocytes and endothelial cells participating in adhesion and transmigration (see Figure B). The PDE8A domains and high intensity staining were seen in both involved and uninvolved UC. In contrast, the control CRC dysplasia showed increased PDE8A staining compared with adjacent non-dysplastic mucosa. A) PDE8A domain visualisation using Image J saturation analysis B) PDE8A domains are present at the site of leukocyte-endothelial cell adhesion (black arrows). Images from involved UC resections of one (A) and three (B) patients. The in situ localisation of PDE8A in UC is consistent with its role in regulating inflammation and adhesion. Together with a favourable therapeutic index, the presence of PDE8A domains at the plasma membrane in involved and uninvolved UC suggests PDE8A is a promising target for anti-adhesion therapy in UC and other immune mediated diseases, perhaps in early disease. Future studies are planned to determine the ability of a PDE8A disruptor to inhibit adhesion molecules in whole blood cultures and intestinal explants. 1. Basole CP, Nguyen RK, Lamothe K, et al. PDE8 controls CD4+ T cell motility through the PDE8A-Raf-1 kinase signaling complex. 2017. http://www.sciencedirect.com/science/article/pii/S0898656817302231

Epitranscriptomic blood biomarkers to manage psychiatric disorders

Major depressive and bipolar disorders are leading causes of disability worldwide yet, many people remain undiagnosed or misdiagnosed or ineffectively treated. Diagnosis relies on the clinical assessment of symptoms and currently, there is no molecular diagnostic test available. Identifying and validating blood biomarkers could provide a more accurate and objective means of diagnosis. Genetic and epigenetic events are involved in psychiatric aetiology, among them RNA editing modifications have been associated with inflammation and neuropsychiatric disorders. Adenosine to inosine RNA editing constitutes a physiological cellular process that translates environmental cues by regulating protein function at the synaptic level in health and disease. RNA editing is post-transcriptional process that leads to functional diversity of proteins. These marks form the molecular interface between the genome and the environment. Of particular interest is the RNA editing modification that occurs on the phosphodiesterase 8A gene located on chromosome 15q25.3, a genomic region that has recurrently been associated with early onset of major depressive disorder. ALCEDIAG???s test, EDITDIAG, identifies in blood specific signatures through the RNA editing modifications of patients in different pathological conditions such as a cohort of hepatitis C infected patients, treated with interferon alpha and ribavirin were followed during 16 weeks. This treatment is well known to trigger depression in 50% of patients. RNA editing modifications were measured each two weeks as well as clinical evaluations of depression (MINI). An algorithm was identified which allows to discriminate patients with depression from others with a high specificity and sensitivity; a cohort of depressed patients (n=163) was compared to controls (n=69). A specific RNA editing signature was identified in depressed patients. The test shows that RNA editing related blood biomarkers allow to stratify patients, characterizes psychiatric conditions and follows up the disease/treatment modifications along time. This test paves the way for a better management of psychiatric patients. Recent Publications 1. Weissmann D, Underwood M, Salvetat N, Cavarec L and Vincent L (2016) Region specific alterations of A-to-I RNA editin of serotonin 2c receptor in cortex of suicides with major depression. Translational Psychiatry 6(8):e878. 2. Van Der Laan S, Salvetat N, Weissmann D and Molina F (2017) Emerging RNA editing biomarkers will foster drug development. Drug Discovery Today 22(7):1056-1063. 3. Cavarec L, Vincent L, Le Borgne C, Plusquellec C and Ollivier N (2013) In Vitro screening for drug-induced depression and/or suicidal adverse effects: a new toxicogenomic assay based on CE-SSCP analysis of HTR2C mRNA editing in SHSY5Y cells. Neurotox Res. 23(1):49-62. 4. Cambon K, Dos-Santos Coura R, Groc L, Carbon A and Weissmann D (2010) Aggressive behavior during social interaction in mice is controlled by the modulation of tyrosine hydroxylase expression in the prefrontal cortex. Neuroscience 171(3):840-51.

Predicting suicidal behavior by an accurate monitoring of RNA editing biomarkers in blood samples

IntroductionPredicting suicidal behaviors is one of the most complex challenges of daily psychiatric practices. Alterations of RNA editing of neurotransmitter receptors and other proteins have been shown to be involved in etiology of different psychiatric disorders and linked to suicidal behavior. Additionally, an increase in expression levels of ADARs, the RNA editing enzymes, has also been observed.ObjectiveThe objective of the present study was to test whether modifications in RNA editing profile of prime targets allow identifying disease-relevant blood biomarkers and evaluating suicide risk in patients.MethodsA clinical study was performed to identify an RNA editing signature in blood of depressed patients with and without history of suicide attempts. Patient's samples were drawn in PAXgene tubes and analyzed on Alcediag's proprietary RNA editing platform using NGS. In addition, gene expression analysis by quantitative PCR was performed.ResultsWe generated a predictive algorithm comprising various selected biomarkers to detect patients with a high risk to attempt suicide. We evaluated the diagnostic performance using the relative proportion of the phosphodiesterase 8A (PDE8A) mRNA editing at different sites as well as the expression of PDE8A and the ADARs. The significance of these biomarkers for suicidality was evaluated using the receiver–operating characteristic (ROC) curve. The generated algorithm comprising the biomarkers was found to have strong diagnostic performances with high specificity and sensitivity.ConclusionsWe developed tools to measure disease–specific biomarkers in blood samples of patients for identifying individuals at the greatest risk for future suicide attempts.Disclosure of interestThe authors have not supplied their declaration of competing interest.

Phosphodiesterase 8a supports HIV-1 replication in macrophages at the level of reverse transcription.

BackgroundHIV-1 infected macrophages play a key role in HIV-1 infection. Even during anti-retroviral treatment, macrophages keep producing virus due to suboptimal tissue penetration and reduced efficacy of antiretrovirals. It is therefore of major importance to understand which host factors are involved in HIV-1 replication in macrophages. Previously, we have shown that genetic polymorphisms in phosphodiesterase 8a (PDE8A) are strongly associated with HIV-1 replication in these cells. Here we analyzed the mechanism and regulation of PDE8A in HIV-1 replication in macrophages.ResultsPDE8A mRNA expression strongly increases upon differentiation of monocytes into macrophages, which corresponds to the increased susceptibility of mature macrophages to HIV-1. In parallel, expression of microRNA miR-145-5p, predicted to target PDE8A mRNA, strongly decreased. The interaction of miR-145-5p with the 3′ UTR of PDE8A mRNA could be experimentally validated, suggesting that indeed miR-145-5p can regulate PDE8A expression levels. Knockdown of PDE8A in macrophages resulted in a decrease in total HIV-1 replication and proviral DNA levels. These observations confirm that PDE8A regulates HIV-1 replication in macrophages and that this effect is mediated through early steps in the viral replication cycle.ConclusionsPDE8A is highly expressed in macrophages, and its expression is regulated by miR-145-5p. Our findings strongly suggest that PDE8A supports HIV-1 replication in macrophages and that this effect is mediated at the level of reverse transcription.

MicroRNA-33a (miR-33a); phosphodiesterase-8A (PDE-8A); UV radiation resistance associated (UVRAG)

MicroRNA-33a (miR-33a); phosphodiesterase-8A (PDE-8A); UV radiation resistance associated (UVRAG)

MiR-33a promotes glioma-initiating cell self-renewal via PKA and NOTCH pathways.

Glioblastoma (GBM) is the most common and lethal brain tumor in adults. Glioma-initiating cells (GICs) are stem-like cells that have been implicated in glioblastoma progression and recurrence; however, the distinct properties of GICs and non-GICs within GBM tumors are largely uncharacterized. Here, we evaluated stem cell-associated microRNA (miR) expression in GICs from GBM patients and GICs derived from xenografted human glioma cell lines and determined that miR-33a promotes GIC growth and self-renewal. Moreover, evaluation of a GBM tissue array revealed that higher miR-33a expression was associated with poor prognosis of GBM patients. Antagonizing miR-33a function in GICs reduced self-renewal and tumor progression in immune-compromised mice, whereas overexpression of miR-33a in non-GICs promoted the display of features associated with GICs. We identified the mRNAs encoding phosphodiesterase 8A (PDE8A) and UV radiation resistance-associated gene (UVRAG) as direct miR-33a targets. PDE8A and UVRAG negatively regulated the cAMP/PKA and NOTCH pathways, respectively; therefore, miR-33a-dependent reduction of these proteins promoted growth and self-renewal of GICs by enhancing PKA and NOTCH activity. Furthermore, in GBM specimens, there was an inverse correlation between the expression levels of miR-33a and PDE8A and UVRAG expression. These findings reveal a miR-33a-centered signaling network that promotes GIC maintenance and has potential as a therapeutic target for GBM treatment.

Phosphodiesterase-8A binds to and regulates Raf-1 kinase

V-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) is a key activator of the ERK pathway and is a target for cross-regulation of this pathway by the cAMP signaling system. The cAMP-activated protein kinase, PKA, inhibits Raf-1 by phosphorylation on S259. Here, we show that the cAMP-degrading phosphodiesterase-8A (PDE8A) associates with Raf-1 to protect it from inhibitory phosphorylation by PKA, thereby enhancing Raf-1's ability to stimulate ERK signaling. PDE8A binds to Raf-1 with high (picomolar) affinity. Mapping of the interaction domain on PDE8A using peptide array technology identified amino acids 454-465 as the main binding site, which could be disrupted by mutation. A cell-permeable peptide corresponding to this region disrupted the PDE8A/Raf-1 interaction in cells, thereby reducing ERK activation and the cellular response to EGF. Overexpression of a catalytically inactive PDE8A in cells displayed a dominant negative phenotype on ERK activation. These effects were recapitulated at the organism level in genetically modified (PDE8A(-/-)) mice. Similarly, PDE8 deletion in Drosophila melanogaster reduced basal ERK activation and sensitized flies to stress-induced death. We propose that PDE8A is a physiological regulator of Raf-1 signaling in some cells.

Transcriptional analysis of rat photoreceptor cells reveals daily regulation of genes important for visual signaling and light damage susceptibility

Photoreceptor cells face the challenge of adjusting their function and, possibly, their susceptibility to light damage to the marked daily changes in ambient light intensity. To achieve a better understanding of photoreceptor adaptation at the transcriptional level, this study aimed to identify genes which are under daily regulation in photoreceptor cells using microarray analysis and quantitative PCR. Included in the gene set obtained were a number of genes which up until now have not been shown to be expressed in photoreceptor cells, such as Atf3 (activating transcription factor 3) and Pde8a (phosphodiesterase 8A), and others with a known impact on phototransduction and/or photoreceptor survival, such as Grk1 (G protein-coupled receptor kinase 1) and Pgc-1α (peroxisome proliferator-activated receptor γ, coactivator 1alpha). According to their daily dynamics, the genes identified could be clustered in two groups: those with peak expression during the second part of the day which are uniformly promoted to cycle by light/dark transitions and those with peak expression during the second part of the night which are predominantly driven by a clock. Since Grk1 and Pgc-1α belong in the first group, the present results support a concept in which transcriptional regulation of genes by ambient light contributes to the functional adjustment of photoreceptor cells over the 24-h period.

CAMP-Specific Phosphodiesterases 8A and 8B, Essential Regulators of Leydig Cell Steroidogenesis

Phosphodiesterase (PDE) 8A and PDE8B are high-affinity, cAMP-specific phosphodiesterases that are highly expressed in Leydig cells. PDE8A is largely associated with mitochondria, whereas PDE8B is broadly distributed in the cytosol. We used a new, PDE8-selective inhibitor, PF-04957325, and genetically ablated PDE8A(-/-), PDE8B(-/-) and PDE8A(-/-)/B(-/-) mice to determine roles for these PDEs in the regulation of testosterone production. PF-04957325 treatment of WT Leydig cells or MA10 cells increased steroid production but had no effect in PDE8A (-/-)/B(-/-) double-knockout cells, confirming the selectivity of the drug. Moreover, under basal conditions, cotreatment with PF-04957325 plus rolipram, a PDE4-selective inhibitor, synergistically potentiated steroid production. These results suggest that the pool(s) of cAMP regulating androgen production are controlled by PDE8s working in conjunction with PDE4. Likewise, PDE8A (-/-)/B(-/-) cells had higher testosterone production than cells from either PDE8A(-/-) or PDE8B(-/-) mice, suggesting that both PDE8s work in concert to regulate steroid production. We further demonstrate that combined inhibition of PDE8s and PDE4 greatly increased PKA activity including phosphorylation of cholesterol-ester hydrolase (CEH)/hormone-sensitive lipase (HSL). CEH/HSL phosphorylation also was increased in PDE8A(-/-)/B(-/-) cells compared with WT cells. Finally, combined inhibition of PDE8s and PDE4 increased the expression of steroidogenic acute regulatory (StAR) protein. Together these findings suggest that both PDE8A and PDE8B play essential roles to maintain low cAMP levels, thereby suppressing resting steroidogenesis by keeping CEH/HSL inactive and StAR protein expression low. They also suggest that in order for PDE inhibitor therapy to be an effective stimulator of steroidogenesis, both PDE8 isozymes and PDE4 need to be simultaneously targeted.

Polymorphism in HIV-1 dependency factor PDE8A affects gene expression and HIV-1 replication in primary macrophages

Background The limited size of the human immunodeficiency virus 1 (HIV-1) genome and the small number of proteins it encodes make the virus highly dependent on host proteins for its replication. Four genome-wide RNAi screens have recently identified a large number of HIV1 dependency factors (HDFs), with the majority of these proteins never before associated with HIV-1 replication. Recently, we reported more than 3 log variation in the ability of HIV-1 to replicate in monocyte derived macrophages (MDM) derived from >4OO healthy seronegative blood donors. In our present study we determined whether single nucleotide polymorphisms (SNPs) in the genes encoding the newly identified HDFs were associated with this variation in HIV-1 replication. Materials and methods DNA from Caucasian donors whose MDM had low (n=96) or high (n=96) viral Gag p24 production, was used for genome-wide SNP genotyping. Linear regression assuming an additive model was used to test for association between the genotype of HDF SNPs and HIV-1 replication in MDM. Results We found a significant association between the minor allele of SNP rs2304418 located in the phosphodiesterase 8a (PDE8A) gene and lower HIV-1 replication (p=2.4×10 -6 ), even after correction for multiple testing. This finding was independent of the CCR5 Δ32 genotype. The minor allele of SNP rs2304418 was also significantly associated with lower PDE8A mRNA levels in MDM (p=8.3×10 -5 )a ndPDE8A mRNA levels correlated with HIV-1 replication. Resequencingof the promotor and untranslated regions of the PDE8A mRNA did not reveal novel SNPs likely to be the causative variant.

Polymorphism in HIV-1 dependency factor PDE8A affects mRNA level and HIV-1 replication in primary macrophages

Four genome-wide RNAi screens have recently identified hundreds of HIV-1 dependency factors (HDFs). Previously, we reported a large variation in the ability of HIV-1 to replicate in monocyte-derived macrophages (MDM) derived from > 400 healthy seronegative blood donors. Here we determined whether SNPs in genes encoding newly identified HDFs were associated with this variation in HIV-1 replication. We found a significant association between the minor allele of SNP rs2304418 in phosphodiesterase 8A ( PDE8A) and lower HIV-1 replication ( p = 2.4 × 10 −6). The minor allele of SNP rs2304418 was also significantly associated with lower PDE8A mRNA levels in MDM ( p = 8.3 × 10 −5). In accordance with this, overexpression of PDE8A in HEK293T cells resulted in increased HIV-1 replication, while subsequent knock-down of PDE8A decreased replication. This study links host genetic variation in a newly identified HDF to variation in HIV-1 replication in a relevant primary target cell for HIV-1 and may provide new leads for treatment of this infection.