Cyclic Phosphodiesterase Research Articles

Antioxidants in health and disease.

Research on antioxidants proceeds at full speed after a partial decline of public interest, when claims on effectiveness of mega doses of vitamin C proved unfounded. The main role of antioxidants is to liquidate the uncontrolled production of reactive oxygen species (ROS) that is being linked to pathogenesis of cardiovascular disease (CVD), malignancy, diabetes type 2, mechanism of infection, fibrogenesis and some neurological disorders. This review summarizes the most recent reports on antioxidants, published since 2010. Follow up data on vitamins C and E focus on their potential for immune modulation and on endothelial nitric oxide bioavailability. Previously well established antioxidants carotenoids and polyphenols are still much in scientific interest. Interestingly, several antioxidants (for example rasveratrol and feruloylnoradrenaline) are generated in infected or injured plants. An intensive attention is directed to resveratrol. This compound, in addition to antioxidation, stimulates nitric oxid production, protects endothelial cells from oxidative functional damage, lowers platelet aggregation and directly inhibits cyclic adenosine monophosphate-specific phosphodiesterases. Recently discovered feruloylnoradrenaline in microbially infected tomatoes is reported to have 14 times the antioxidant power of resveratrol. With all this new information, it is important to point out the prevailing opinion that additional supplements of antioxidants are not needed, as long as the organism has adequate stores of antioxidants and the diet is normal in composition and in quantity (Fig. 3, Ref. 34).

Comparative proteomic analysis of midgut proteins from male and female Bombyx mori (Lepidoptera: Bombycidae).

Many biological phenotypes of male and female silkworms ( Bombyx mori ) are quite different, and one of the major differences is the growth rate at various larval stages. Nutrient utilization by midgut varies with sexes. However, the molecular basis of this variation is not clear. To understand the molecular mechanism, comparative proteomic approach was employed to investigate the variation of midgut proteomes between male and female silkworms. Totally, 32 proteins that were grouped into four categories were differentially expressed and subsequently identified by mass spectrometry. Gene ontology analysis revealed that these proteins were attributed with biological functions such as binding, catalytic, and transporter, and these proteins were involved in biological process such as cellular process, localization, and metabolic process. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these proteins were involved in pathways such as glycolysis, gluconeogenesis, oxidative phosphorylation, and purine metabolism. At transcription level, the expressional variation was confirmed for six identified proteins including muscle glycogen phosphorylase, uridine 5'-monophosphate synthase, cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha, ATP synthase, thiol peroxiredoxin, and serpin-2. This study provides useful information for understanding the mechanisms of nutrient absorption and the protein–protein interaction in the silkworm.

A novel trinuclear iron center is revealed by the structure of a cyclic di-GMP HD-GYP domain phosphodiesterase from Persephonella marina. A detailed view of the HD-GYP domain of P. marina is shown in ribbon presentation with iron atoms in orange and key m

Molecular MicrobiologyVolume 91, Issue 1 p. i-i Front CoverFree Access A novel trinuclear iron center is revealed by the structure of a cyclic di-GMP HD-GYP domain phosphodiesterase from Persephonella marina. A detailed view of the HD-GYP domain of P. marina is shown in ribbon presentation with iron atoms in orange and key metal binding residues His221 and Asp222 in stick format. Also shown are the residues of the GYP motif. For further details readers are referred to the article by Bellini et al. on pp. 26-38 of this issue. First published: 27 December 2013 https://doi.org/10.1111/mmi.12494AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat No abstract is available for this article. Volume91, Issue1January 2014Pages i-i RelatedInformation

LTQ-XL mass spectrometry proteome analysis expands the Pseudomonas aeruginosa AmpR regulon to include cyclic di-GMP phosphodiesterases and phosphoproteins, and identifies novel open reading frames

The AmpR proteome data not only confirmed the role of AmpR in virulence and resistance to multiple antibiotics, but also expanded the perimeter of AmpR regulon. The data presented here points to the role of AmpR in regulating cyclic di-GMP levels and phosphorylation of Ser, Thr and Tyr, adding another dimension to the regulatory functions of AmpR. We also identify some previously unannotated/misannotated ORFs in the P. aeruginosa genome, indicating the limitations of existing ORF analyses software. This study will contribute towards understanding complex genetic organization of P. aeruginosa. Whole genome proteomic picture of regulators at higher nodal positions in the regulatory network will not only help us link various virulence phenotypes but also design novel therapeutic strategies.

Effects of Water-Soluble Polysubstituted Fullerene Derivatives on Sarcoplasmic Reticulum Ca2+-ATPase and Cyclic Guanosine Monophosphate Phosphodiesterase Activities

The effects of new fullerene derivatives on the functioning of the hydrolases sarcoplasmic reticulum (SR) Ca2+-ATPase and cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) were studied. Most of the fullerene derivatives studied were found to inhibit the activity of both enzymes. Thus, compounds PSF-1, PSF-II, and PSF-III completely inhibited Ca2+ transport at a concentration of 0.009 mM but inhibited ATP hydrolysis by only 55 ± 6%, 42 ± 8%, and 60 ± 6% respectively, thus uncoupling the hydrolytic and transport functions of the enzyme. This induced changes in the extra- and intracellular Ca2+ ion levels and affected adhesion of metastatic cells to the capillary endothelium. Compound PSF-III had noncompetitive, reversible effects on the hydrolytic function of SR Ca2+-ATPase with K i = 1.6 × 10–6 M. All the fullerene derivatives studied inhibited cGMP PDE function and could induce an antiaggregation effect which resulted from accumulation of the cGMP PDE substrate, i.e., cGMP, which is a second messenger in living organisms. These results can be used to predict the potential therapeutic effects of the fullerene derivatives studied.

Discovery of a Cyclic Phosphodiesterase That Catalyzes the Sequential Hydrolysis of Both Ester Bonds to Phosphorus

The bacterial C-P lyase pathway is responsible for the metabolism of unactivated organophosphonates under conditions of phosphate starvation. The cleavage of the C-P bond within ribose-1-methylphosphonate-5-phosphate to form methane and 5-phospho-ribose-1,2-cyclic phosphate (PRcP) is catalyzed by the radical SAM enzyme PhnJ. In Escherichia coli the cyclic phosphate product is hydrolyzed to ribose-1,5-bisphosphate by PhnP. In this study, we describe the discovery and characterization of an enzyme that can hydrolyze a cyclic phosphodiester directly to a vicinal diol and inorganic phosphate. With PRcP, this enzyme hydrolyzes the phosphate ester at carbon-1 of the ribose moiety to form ribose-2,5-bisphosphate, and then this intermediate is hydrolyzed to ribose-5-phosphate and inorganic phosphate. Ribose-1,5-bisphosphate is neither an intermediate nor a substrate for this enzyme. Orthologues of this enzyme are found in the human pathogens Clostridium difficile and Eggerthella lenta. We propose that this enzyme be called cyclic phosphate dihydrolase (cPDH) and be designated as PhnPP.

Changes in Energy-Regulated Molecules in the Trophocytes and Fat Cells of Young and Old Worker Honeybees (Apis mellifera)

Trophocytes and fat cells of honeybees (Apis mellifera) have been used for cellular senescence studies, but the changes in the expression, concentration, and activity of cellular energy-regulated molecules that occur with aging in worker bees is unknown. In this study, energy-regulated molecules were evaluated in the trophocytes and fat cells of young and old workers. The results showed that (i) adenosine monophosphate-activated protein kinase-α2 (AMPK-α2) expression increased with aging, whereas phosphorylated AMPK-α2 expression, the phosphorylated AMPK/AMPK ratio, and AMPK activity decreased with aging; (ii) adenosine diphosphate and adenosine triphosphate concentrations decreased with aging, the AMP concentration was unchanged, the adenosine diphosphate/adenosine triphosphate ratio did not change with aging, and the AMP/adenosine triphosphate ratio increased with aging; (iii) the cyclic AMP concentration decreased with aging, and cyclic AMP-specific phosphodiesterases activity increased with aging; (iv) silent information regulator 2 (Sir2) expression increased with aging, whereas its activity decreased with aging; and (v) peroxisome proliferator-activated receptor-α expression decreased with aging. These results show that the trophocytes and fat cells of young workers have higher cellular energy status and express higher levels of energy-regulated molecules than those of old workers and that aging results in a decline in the energy status of trophocytes and fat cells in worker honeybees.

Evaluation of Novel Acyclic Nucleoside Phosphonates against Human and Animal Gammaherpesviruses Revealed an Altered Metabolism of Cyclic Prodrugs upon Epstein-Barr Virus Reactivation in P3HR-1 Cells

Acyclic nucleoside phosphonates (ANPs), such as (S)-1-[(3-hydroxy-2-phosphonomethoxy)propyl)]cytosine (HPMPC), are an important group of broad-spectrum antiviral agents with activity against DNA viruses. In this report, we present the in vitro potencies of novel ANPs against gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus, Epstein-Barr virus (EBV), and three animal gammaherpesviruses. 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC), (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]-3-deazaadenine (3-deaza-HPMPA), and their cyclic derivatives have emerged as highly potent antigammaherpesvirus agents. Interestingly, cyclic prodrugs of ANPs exhibited reduced activities against EBV strain P3HR-1, but not against EBV strain Akata. Cell culture metabolism studies with HPMPC and cyclic HPMPC revealed that these differences were attributable to an altered drug metabolism in P3HR-1 cells after EBV reactivation and, more specifically, to a reduced hydrolysis of cyclic HPMPC by cyclic CMP phosphodiesterase. We did not correlate this effect with phosphodiesterase downregulation, or to functional mutations. Instead, altered cyclic AMP levels in P3HR-1 cells indicated a competitive inhibition of the phosphodiesterase by this cyclic nucleotide. Finally, both HPMPC and HPMP-5-azaC emerged as highly effective inhibitors in vivo through significant inhibition of murine gammaherpesvirus replication and dissemination. With the current need for potent antigammaherpesvirus agents, our findings underline the requirement of appropriate surrogate viruses for antiviral susceptibility testing and highlight HPMP-5-azaC as a promising compound for future clinical development.

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.

Effect of Cyclic Hydroxamic Acids Derived from Glycine and D,L-Alanine on Activity of Ca2+, Mg2+-ATPase Hydrolases of Sarcoplasmic Reticulum and Cyclic Guanosine Monophosphate Phosphodiesterase

The effects of cyclic hydroxamic acids (CHAs) derived from glycine and D,L-alanine on the enzymatic activity of Ca2+,Mg2+-ATPase from sarcoplasmic reticulum (Ca2+,Mg2+-ATPase SR) and cyclic guanosine monophosphate phosphodiesterase (PDEcGMP) were investigated. CHAs I (C5H10N2O2), II (C6H12N2O2), III (C8H15N3O2), IV (C9H17N3O2), V (C11H21N3O2), and VI (C12H23N3O2) were modulators of Ca2+,Mg2+-ATPase SR enzyme activity. Compounds I-VI decoupled to various extents the hydrolytic and transport functions of Ca2+,Mg2+-ATPase SR, disrupting the ratio of intra- and extracellular Ca2+ ions. This affected the adhesion of metastatic cells to capillary endothelium. Compounds IV and VI had the highest metastasis inhibition indices (MII%) for B-16 melanoma of 33 and 81%, respectively. This correlated with a decreased Ca2+ transmembrane transfer coefficient into SR vesicles of 0.75 for IV and 0.5 for VI compared with a [Ca2+]/[ATP] ratio in the control of 1.4. CHAs I-VI did not affect the functioning of PDEcGMP. The results enabled potential antimetastatic drugs in the CHA series to be predicted.

An HD-GYP Cyclic Di-Guanosine Monophosphate Phosphodiesterase with a Non-Heme Diiron–Carboxylate Active Site

The intracellular level of the ubiquitous bacterial secondary messenger, cyclic di-(3',5')-guanosine monophosphate (c-di-GMP), represents a balance between its biosynthesis and degradation, the latter via specific phosphodiesterases (PDEs). One class of c-di-GMP PDEs contains a characteristic HD-GYP domain. Here we report that an HD-GYP PDE from Vibrio cholerae contains a non-heme diiron-carboxylate active site, and that only the reduced form is active. An engineered D-to-A substitution in the HD dyad caused loss of c-di-GMP PDE activity and of two iron atoms. This report constitutes the first demonstration that a non-heme diiron-carboxylate active site can catalyze the c-di-GMP PDE reaction and that this activity can be redox regulated in the HD-GYP class.

Cyclic di-AMP Is Critical for Listeria monocytogenes Growth, Cell Wall Homeostasis, and Establishment of Infection

ABSTRACTListeria monocytogenes infection leads to robust induction of an innate immune signaling pathway referred to as the cytosolic surveillance pathway (CSP), characterized by expression of beta interferon (IFN-β) and coregulated genes. We previously identified the IFN-β stimulatory ligand as secreted cyclic di-AMP. Synthesis of c-di-AMP in L. monocytogenes is catalyzed by the diadenylate cyclase DacA, and multidrug resistance transporters are necessary for secretion. To identify additional bacterial factors involved in L. monocytogenes detection by the CSP, we performed a forward genetic screen for mutants that induced altered levels of IFN-β. One mutant that stimulated elevated levels of IFN-β harbored a transposon insertion in the gene lmo0052. Lmo0052, renamed here PdeA, has homology to a cyclic di-AMP phosphodiesterase, GdpP (formerly YybT), of Bacillus subtilis and is able to degrade c-di-AMP to the linear dinucleotide pApA. Reduction of c-di-AMP levels by conditional depletion of the di-adenylate cyclase DacA or overexpression of PdeA led to marked decreases in growth rates, both in vitro and in macrophages. Additionally, mutants with altered levels of c-di-AMP had different susceptibilities to peptidoglycan-targeting antibiotics, suggesting that the molecule may be involved in regulating cell wall homeostasis. During intracellular infection, increases in c-di-AMP production led to hyperactivation of the CSP. Conditional depletion of dacA also led to increased IFN-β expression and a concomitant increase in host cell pyroptosis, a result of increased bacteriolysis and subsequent bacterial DNA release. These data suggest that c-di-AMP coordinates bacterial growth, cell wall stability, and responses to stress and plays a crucial role in the establishment of bacterial infection.

Abstract 3659: Next generation sulindac.

Abstract Sulindac displays promising antineoplastic activity, but toxicity from cyclooxygenase (COX) inhibition limits its long-term use for chemoprevention. Previous studies have concluded that the tumor cell growth inhibitory activity of sulindac is COX-independent and suggest that safer and more efficacious drugs can be developed by targeting the underlying mechanism. Here we report that sulindac sulfide (SS) inhibits cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) activity, increases intracellular cGMP levels and activates protein kinase G at concentrations that suppress proliferation and induce apoptosis of colon tumor cells. Normal colonocytes were refractory to the anti-proliferative and pro-apoptotic effects of SS as well as its ability to induce cGMP signaling. PDE5-specific inhibitors such as tadalafil also inhibited the growth of colon tumor cells that expressed high levels of the cGMP-specific PDE5 isozyme compared with colonocytes. RNAi knockdown of PDE5 selectively inhibited proliferation and induced apoptosis of colon tumor cells as did SS. SS, 8-bromo-cGMP, and PDE5 siRNA reduced β-catenin levels, as well as the expression of cyclin D and survivin that are regulated by β-catenin-dependent TCF transcriptional activity by a mechanism involving the transcriptional repression of β-catenin. The significance of these findings are highlighted by studies in the FCCC Min mouse model of intestinal tumorigenesis showing that a novel non-COX inhibitory derivative of SS can strongly inhibit colon tumor formation by a mechanism that appears to involve cGMP PDE inhibition. These observations suggest that safer and more efficacious drugs can be developed for colorectal cancer chemoprevention by targeting PDE5 and possibly other cGMP degrading isozymes. Citation Format: Nan Li, Yaguang Xi, Heather N. Tinsley, Evrim Gurpinar, Bernard D. Gary, Bing Zhu, Yonghe Li, Adam B. Keeton, William E. Grizzle, Gary A. Piazza. Next generation sulindac. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3659. doi:10.1158/1538-7445.AM2013-3659

Phenotype overlap in Xylella fastidiosa is controlled by the cyclic di-GMP phosphodiesterase Eal in response to antibiotic exposure and diffusible signal factor-mediated cell-cell signaling.

Eal is an EAL domain protein in Xylella fastidiosa homologous to one involved in resistance to tobramycin in Pseudomonas aeruginosa. EAL and HD-GYP domain proteins are implicated in the hydrolysis of the secondary messenger bis-(3'-5')-cyclic dimeric GMP (cyclic di-GMP). Cell density-dependent communication mediated by a Diffusible Signal Factor (DSF) also modulates cyclic di-GMP levels in X. fastidiosa, thereby controlling the expression of virulence genes and genes involved in insect transmission. The possible linkage of Eal to both extrinsic factors such as antibiotics and intrinsic factors such as quorum sensing, and whether both affect virulence, was thus addressed. Expression of eal was induced by subinhibitory concentrations of tobramycin, and an eal deletion mutant was more susceptible to this antibiotic than the wild-type strain and exhibited phenotypes similar to those of an rpfF deletion mutant blocked in DSF production, such as hypermotility, reduced biofilm formation, and hypervirulence to grape. Consistent with that, the rpfF mutant was more susceptible than the wild-type strain to tobramycin. Therefore, we propose that cell-cell communication and antibiotic stress can apparently lead to similar modulations of cyclic di-GMP in X. fastidiosa, resulting in similar phenotypes. However, the effect of cell density is dominant compared to that of antibiotic stress, since eal is suppressed by RpfF, which may prevent inappropriate behavioral changes in response to antibiotic stress when DSF accumulates.

Gene Cloning, Expression, and Characterization of a Cyclic Nucleotide Phosphodiesterase from Arthrobacter sp. CGMCC 3584

Based on thermal asymmetric interlaced polymerase chain reaction, the arpde gene encoding a cyclic nucleotide-specific phosphodiesterase was cloned from Arthrobacter sp. CGMCC 3584 for the first time. The 930-bp region encoded a 309-amino-acid protein with a molecular weight of 33.6 kDa. The recombinant ArPDE was able to hydrolyze 3',5'-cAMP, 3',5'-cGMP, and 2',3'-cAMP. The K m values of ArPDE for 3',5'-cAMP and 3',5'-cGMP were 6.82 and 12.82 mM, respectively. ArPDE was thermostable and displayed optimal activity at 45 °C and pH 7.5. The enzyme did not require any metal cofactors, although its activity was stimulated by 2 mM Co(2+) and inhibited by Zn(2+). Nucleotides, reducing agents, and sulfhydryl reagents had different inhibitory effects on the activity of ArPDE. NaF, the actual compound used to improve the industrial yield of cAMP, exhibited 62 % inhibitions at concentrations of 10 mM.

Host Cell Contact Induces Expression of Virulence Factors and VieA, a Cyclic di-GMP Phosphodiesterase, in Vibrio cholerae

Vibrio cholerae, a noninvasive bacterium, colonizes the intestinal epithelium and secretes cholera toxin (CT), a potent enterotoxin that causes the severe fluid loss characteristic of the disease cholera. In this study, we demonstrate that adherence of V. cholerae to the intestinal epithelial cell line INT 407 strongly induces the expression of the major virulence genes ctxAB and tcpA and the virulence regulatory gene toxT. No induction of toxR and tcpP, which encode transcriptional activators of toxT, was observed in adhered bacteria, and the adherence-dependent upregulation of toxT expression was independent of ToxR and TcpP. A sharp increase in the expression of the vieA gene, which encodes a cyclic di-GMP (c-di-GMP) phosphodiesterase, was observed in INT 407-adhered V. cholerae immediately after infection. Induction of toxT, ctxAB, and tcpA in INT 407-adhered vieA mutant strain O395 ΔvieA was consistently lower than in the parent strain, although no effect was observed in unadhered bacteria, suggesting that VieA has a role in the upregulation of toxT expression specifically in host cell-adhered V. cholerae. Furthermore, though VieA has both a DNA binding helix-turn-helix domain and an EAL domain conferring c-di-GMP phosphodiesterase activity, the c-di-GMP phosphodiesterase activity of VieA is necessary and sufficient for the upregulation of toxT expression.

Functional Proteomic Profiling of Phosphodiesterases Using SeraFILE Separations Platform

Functional proteomic profiling can help identify targets for disease diagnosis and therapy. Available methods are limited by the inability to profile many functional properties measured by enzymes kinetics. The functional proteomic profiling approach proposed here seeks to overcome such limitations. It begins with surface-based proteome separations of tissue/cell-line extracts, using SeraFILE, a proprietary protein separations platform. Enzyme kinetic properties of resulting subproteomes are then characterized, and the data integrated into proteomic profiles. As a model, SeraFILE-derived subproteomes of cyclic nucleotide-hydrolyzing phosphodiesterases (PDEs) from bovine brain homogenate (BBH) and rat brain homogenate (RBH) were characterized for cAMP hydrolysis activity in the presence (challenge condition) and absence of cGMP. Functional profiles of RBH and BBH were compiled from the enzyme activity response to the challenge condition in each of the respective subproteomes. Intersample analysis showed that comparable profiles differed in only a few data points, and that distinctive subproteomes can be generated from comparable tissue samples from different animals. These results demonstrate that the proposed methods provide a means to simplify intersample differences, and to localize proteins attributable to sample-specific responses. It can be potentially applied for disease and nondisease sample comparison in biomarker discovery and drug discovery profiling.

Molecule of the month HIV-1 Tat and miRNA.

miRNAs are a well-established RNA phenomenon derived from the non-coding major portion of the human genome. miRNAs cannot be ignored because they are so heavily involved in the control of gene expression [1]. The study of neurological and neuropsychiatric disease also has miRNA components that are commencing to be mapped and characterized. Due to their high stability, for example miRNA can be found in cerebrospinal fluid (CSF) as well as within cells. In a recent publication, comparisons of miRNAs were made among individuals that were HIV positive, without and with HIV encephalitis (HIVE), in individuals with HIV-Associated Neurological Disorders (HAND), and HIV negative controls. CSF and frontal cortex were studied. Comparing HIV positives with HIV negatives, a total of 66 miRNAs were found differentially upregulated for HIV positive individuals. The HIV negative cases were a type of disease control in that they had non-viral acute encephalomyelitis. Of the 66 upregulated miRNAs, statistical analyses demonstrated that 11 miRNAs were upregulated significantly [2]. HIV-1 Tat is an important virus coded regulator protein produced during HIV-1 infection [3]. HIV-1 Tat disrupts neuronal function via miRNAs. Specifically, miRNA expression becomes altered in neuron cultures exposed to HIV-1 tat. Tat is a high inducer of miR-34a as well as other miRNAs. However, tat also decreased levels of target genes including CREB involving miR-34a [4]. Earlier studies on the effects of Tat specifically and HIV-1 in general need to be reinterpreted in terms of the involvement of miRNAs. For example, PC12 (neuronal) cell cultures demonstrate a rapid (ca. 5 minutes) increase in cAMP and CREB transcription factor levels upon exposure to HIV-1 Tat. The serine-133 (Ser-133) amino acid is phosphorylated on CREB. However, when this experiment is done at later times of addition of Tat (ca. 1-2 hours), there is a continued decline of cAMP and reduction of phosphorylated CREB. The dephosphorylation was shown to be due to activation of a phosphatidyl-inositol-3-kinase, AKT, and cyclic nucleoside phosphodiesterase pathway. In addition, there was increased apoptosis of the treated cells. These findings may be relevant for the mechanisms of pathogenesis for HIV-1-associated dementia (HAD) [5] as well as HAND. In related studies, HIV-1 Tat protein was shown to affect histone deacetylases (HDACs) (that are central in transcriptional epigenetics and remodeling of chromatin). Tat protein upregulates HDAC2 in neuronal cells. This may result in inhibition of transcription in neurons and down regulation for Ca2+/calmodulin-dependent protein kinase II (CaMKII) as well as CREB gene expression. These genes are involved in neuronal function and synaptic plasticity. Moreover, as indicated by findings described above, miRNAs may be involved. These pathways may be components of dysfunction that lead to HAD and HAND. Consequently, specific HDAC2 inhibitors may be of use in NeuroAIDS [6].The manipulation of miRNA expression may become an important component of the attack on disease. The figure illustrates various gene interactions. It is left as a puzzle for the interested reader to identify the various genes and their functions in the (Figure 1) [7, 8]. Figure 1 CREB HDAC2 CaMKII Network. The graph shows interactions among proteins CREB, HDAC2, and CaMKII. As mentioned in the text, the expression of these proteins may be modulated by miRNAs that are consequent to HIV-1 infection. In this figure, line-colors and ...

Abstract B59: Cyclic GMP phosphodiesterase: A molecular target for cancer chemoprevention

Abstract Nonsteroidal anti-inflammatory drugs (NSAIDs) can significantly reduce the incidence and risk of death from multiple cancers, but their long-term use for chemoprevention is not recommended because of potentially fatal toxicities resulting from cyclooxygenase (COX) inhibition. However, the mechanism responsible for their tumor cell growth inhibitory activity may not require COX inhibition, which suggests that NSAID derivatives lacking COX inhibitory activity have potential for improved safety and efficacy. Cyclic guanosine monophosphate phosphodiesterase (cGMP PDE) inhibition appears to be an important mechanism based on previous studies with the NSAID, sulindac. Here we characterize a novel sulindac derivative referred to as sulindac benzylamine (SBA) that does not inhibit COX-1 or COX-2, yet potently inhibits the growth and induces the apoptosis of human colon tumor cells. This activity is closely associated with cGMP PDE inhibition given that SBA can inhibit cGMP hydrolysis in colon tumor cell lysates, increase intracellular cGMP levels, and activate cGMP-dependent protein kinase G (PKG) at concentrations that suppress tumor cell growth and induce apoptosis. The cGMP-specific PDE isozyme, PDE5 is selectively inhibited by SBA and elevated in colon tumor cells compared with normal colonocytes that display reduced sensitivity to SBA. PKG activation by SBA is associated with the reduction of nuclear β-catenin levels and inhibition of Tcf transcriptional activity in which the anti-apoptosis gene, survivin is suppressed. These events preceded apoptosis induction by SBA and appear to result from the rapid elevation of intracellular cGMP levels following cGMP PDE inhibition. Transient transfection with PDE5 siRNA reduced PDE5 protein expression in both human normal (NCM460) colonocytes and colon tumor cell lines (HT29 and HCT116), but only suppressed cGMP PDE activity in the tumor cells, likely because of low PDE5 levels and/or the presence of other cGMP degrading isozymes in normal colonocytes. In accord, PDE5 siRNA selectively suppressed growth and induced apoptosis of colon tumor cells, but not normal colonocytes. Colon tumor cells transfected with PDE5 siRNA also displayed reduced β-catenin levels and Tcf transcriptional activity as well as increased sensitivity to SBA treatment. Finally, molecular modeling studies suggest that SBA directly interferes with cGMP binding within the catalytic domain of PDE5. However, additional cGMP degrading PDE isozymes may either be responsible for or contribute to the sensitivity of tumor cells to SBA given that PDE5 selective inhibitors (e.g. sildenafil) do not effectively suppress colon tumor cell growth. These results lead us to conclude that cGMP PDE inhibition is an important molecular target for cancer chemoprevention, although further studies are necessary to identify key cGMP degrading isozymes involved in tumorigenesis. This research was supported NIH grants, NCI 1R01CA131378 and 1R01CA148817-01A1. Citation Format: Gary A. Piazza, Nan Li, Gary D. Bernard, Xi Chen, Adam B. Keeton, Yaguang Xi, Bing Zhu, Wei Zhang, Alexandra Fajardo, Veronica Ramirez, Sara Sigler, Kevin Lee, Claire Cawthon, Evrim Gurpinar, Maya A. Brownby, Heather N. Tinsely, Ashraf Abadi, William Grizzle. Cyclic GMP phosphodiesterase: A molecular target for cancer chemoprevention. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B59.

Selective PDE5A inhibition with sildenafil rescues left ventricular dysfunction, inflammatory immune response and cardiac remodeling in angiotensin II-induced heart failure in vivo

Sildenafil inhibits cyclic GMP-specific phosphodiesterase type-5A (PDE5A) and can prevent cardiac hypertrophy and left ventricular (LV) dysfunction in mice subjected to severe pressure-overload. The pathophysiological role of sildenafil in adverse remodeling in the hypertensive heart after chronic renin-angiotensin aldosterone system stimulation is unknown. Therefore, we studied the efficacy of the PDE5A inhibitor sildenafil for treating advanced cardiac hypertrophy and LV remodeling due to angiotensin (Ang)II-induced heart failure (HF) in vivo. C57BL6/J mice were subjected to AngII-induced cardiac hypertrophy for 3weeks and cardiac dysfunction, cardiac inflammatory stress response, adverse remodeling as well as apoptosis were documented. Mice were subsequently treated with sildenafil (100mg/kg/day) or placebo with delay of 5days for treating AngII infusion-induced adverse events. Compared to controls, AngII infusion resulted in impaired systolic (dP/dt (max) -46%, SV -16%, SW -43%, E (a) +51%, EF -37%, CO -36%; p<0.05) and diastolic (dP/dt (min) -36%, LV end diastolic pressure +73%, Tau +21%, stiffness constant β +74%; p<0.05) LV function. This was associated with a significant increase in cardiac hypertrophy and fibrosis. Increased inflammatory response was also indicated by an increase in immune cell infiltration and apoptosis. Treatment with sildenafil led to a significant improvement in systolic and diastolic LV performance. This effect was associated with less LV hypertrophy, remodeling, cardiac inflammation and apoptosis. PDE5A inhibition with sildenafil may provide a new treatment strategy for cardiac hypertrophy and adverse remodeling in the hypertensive heart.