Phosphodiesterase 9A controls nitric-oxide-independent cGMP and hypertrophic heart disease.

Cyclic guanosine monophosphate (cGMP) is a second messenger molecule that transduces nitric-oxide- and natriuretic-peptide-coupled signalling, stimulating phosphorylation changes by protein kinase G. Enhancing cGMP synthesis or blocking its degradation by phosphodiesterase type 5A (PDE5A) protects against cardiovascular disease. However, cGMP stimulation alone is limited by counter-adaptions including PDE upregulation. Furthermore, although PDE5A regulates nitric-oxide-generated cGMP, nitric oxide signalling is often depressed by heart disease. PDEs controlling natriuretic-peptide-coupled cGMP remain uncertain. Here we show that cGMP-selective PDE9A (refs 7, 8) is expressed in the mammalian heart, including humans, and is upregulated by hypertrophy and cardiac failure. PDE9A regulates natriuretic-peptide- rather than nitric-oxide-stimulated cGMP in heart myocytes and muscle, and its genetic or selective pharmacological inhibition protects against pathological responses to neurohormones, and sustained pressure-overload stress. PDE9A inhibition reverses pre-established heart disease independent of nitric oxide synthase (NOS) activity, whereas PDE5A inhibition requires active NOS. Transcription factor activation and phosphoproteome analyses of myocytes with each PDE selectively inhibited reveals substantial differential targeting, with phosphorylation changes from PDE5A inhibition being more sensitive to NOS activation. Thus, unlike PDE5A, PDE9A can regulate cGMP signalling independent of the nitric oxide pathway, and its role in stress-induced heart disease suggests potential as a therapeutic target.

Increased Intracellular Cyclic di-AMP Levels Sensitize Streptococcus gallolyticus subsp. gallolyticus to Osmotic Stress and Reduce Biofilm Formation and Adherence on Intestinal Cells.

Cyclic di-AMP is a recently identified second messenger exploited by a number of Gram-positive bacteria to regulate important biological processes. Here, we studied the phenotypic alterations induced by the increased intracellular c-di-AMP levels in Streptococcus gallolyticus, an opportunistic pathogen responsible for septicemia and endocarditis in the elderly. We report that an S. gallolyticus c-di-AMP phosphodiesterase gdpP knockout mutant, which displays a 1.5-fold higher intracellular c-di-AMP levels than the parental strain UCN34, is more sensitive to osmotic stress and is morphologically smaller than the parental strain. Unexpectedly, we found that a higher level of c-di-AMP reduced biofilm formation of S. gallolyticus on abiotic surfaces and reduced adherence and cell aggregation on human intestinal cells. A genome-wide transcriptomic analysis indicated that c-di-AMP regulates many biological processes in S. gallolyticus, including the expression of various ABC transporters and disease-associated genes encoding bacteriocin and Pil3 pilus. Complementation of the gdpP in-frame deletion mutant with a plasmid carrying gdpP in trans from its native promoter restored bacterial morphology, tolerance to osmotic stress, biofilm formation, adherence to intestinal cells, bacteriocin production, and Pil3 pilus expression. Our results indicate that c-di-AMP is a pleiotropic signaling molecule in S. gallolyticus that may be important for S. gallolyticus pathogenesis.IMPORTANCEStreptococcus gallolyticus is an opportunistic pathogen responsible for septicemia and endocarditis in the elderly and is also strongly associated with colorectal cancer. S. gallolyticus can form biofilms, express specific pili to colonize the host tissues, and produce a specific bacteriocin allowing killing of commensal bacteria in the murine colon. Nevertheless, how the expression of these colonization factors is regulated remains largely unknown. Here, we show that c-di-AMP plays pleiotropic roles in S. gallolyticus, controlling the tolerance to osmotic stress, cell size, biofilm formation on abiotic surfaces, adherence and cell aggregation on human intestinal cells, expression of Pil3 pilus, and production of bacteriocin. This study indicates that c-di-AMP may constitute a key regulatory molecule for S. gallolyticus host colonization and pathogenesis.

The high-affinity cAMP-specific phosphodiesterase 8B controls steroidogenesis in the mouse adrenal gland.

The functions of the phosphodiesterase 8B (PDE8) family of phosphodiesterases have been largely unexplored because of the unavailability of selective pharmacological inhibitors. Here, we report a novel function of PDE8B as a major regulator of adrenal steroidogenesis using a genetically ablated PDE8B mouse model as well as cell lines treated with either a new PDE8-selective inhibitor or a short hairpin RNA (shRNA) construct against PDE8B. We demonstrate that PDE8B is highly enriched in mouse adrenal fasciculata cells, and show that PDE8B knockout mice have elevated urinary corticosterone as a result of adrenal hypersensitivity toward adrenocorticotropin. Likewise, ablation of PDE8B mRNA transcripts by an shRNA construct potentiates steroidogenesis in the commonly used Y-1 adrenal cell line. We also observed that the PDE8-selective inhibitor (PF-04957325) potentiates adrenocorticotropin stimulation of steroidogenesis by increasing cAMP-dependent protein kinase activity in both primary isolated adrenocortical cells and Y-1 cells. It is noteworthy that PDE8s have their greatest control under low adrenocorticotropin-stimulated conditions, whereas other higher K(m) PDE(s) modulate steroidogenesis more effectively when cells are fully stimulated. Finally, both genetic ablation of PDE8B and long-term pharmacological inhibition of PDE8s cause increased expression of steroidogenic enzymes. We conclude that PDE8B is a major regulator of one or more pools of cAMP that promote steroidogenesis via both short- and long-term mechanisms. These findings further suggest PDE8B as a potential therapeutic target for the treatment of several different adrenal diseases.

PDE9A deficiency does not prevent chronic-hypoxic pulmonary hypertension in mice.

Inhibition of cyclic guanosine monophosphate (cGMP)-specific phosphodiesterases (PDEs) is a cornerstone of pulmonary arterial hypertension (PAH)-specific therapy. PDE9A, expressed in the heart and lung tissue, has the highest affinity for cGMP of all known PDEs. PDE9A deficiency protects mice against chronic left ventricular (LV) pressure overload via increased natriuretic peptide (NP)-dependent cGMP signaling. Chronic-hypoxic pulmonary hypertension (CH-PH) is a model of chronic right ventricular (RV) pressure overload, and previous studies have demonstrated a protective role for NPs in the murine model. Therefore, we hypothesized that PDE9A deficiency would promote NP-dependent cGMP signaling and prevent RV remodeling in the CH-PH model, analogous to findings in the LV. We exposed wild-type and PDE9A-deficient (Pde9a-/- ) C57BL/6 mice to CH-PH for 3 weeks. We measured RV pressure, hypertrophy, and levels of lung and RV cGMP, PDE9A, PDE5A, and phosphorylation of the protein kinase G substrate VASP (vasodilatory-stimulated phosphoprotein) after CH-PH. In wild-type mice, CH-PH was associated with increased circulating ANP and lung PDE5A, but no increase in cGMP, PDE9A, or VASP phosphorylation. Downstream effectors of cGMP were not increased in Pde9a-/- mice exposed to CH-PH compared with Pde9a+/+ littermates, and CH-PH induced increases in RV pressure and hypertrophy were not attenuated in knockout mice. Taken together, these findings argue against a prominent role for PDE9A in the murine CH-PH model.

Polyphosphate kinase affects oxidative stress response by modulating cAMP receptor protein and rpoS expression in Salmonella typhimurium.

Polyphosphate (polyP) plays diverse physiological functions in prokaryotes and eukaryotes, but most of their detailed mechanisms are still obscure. Here, we show that deletion of polyphosphate kinase (PPK), the principal enzyme responsible for synthesis of polyP, resulted in augmented expression of cAMP receptor protein (CRP) and rpoS and lowered H2O2 sensitivity in Salmonella Typhimurium ATCC14028. The binding of cAMP-CRP complex to rpoS promoter and further stimulation of its transcription were proved through electrophoretic mobility shift assay, lacZ fusion, and exogenous cAMP addition, respectively. The rpoS expression increased in cpdA (cAMP phosphodiesterase coding gene) mutant, further suggesting that cAMP-CRP upregulated rpoS expression. These results demonstrate that PPK affects oxidative stress response by modulating crp and rpoS expression in S. Typhimurium.

Oscillatory cAMP cell-cell signalling persists during multicellular Dictyostelium development.

Propagating waves of cAMP, periodically initiated in the aggregation centre, are known to guide the chemotactic aggregation of hundreds of thousands of starving individual Dictyostelium discoideum cells into multicellular aggregates. Propagating optical density waves, reflecting cell periodic movement, have previously been shown to exist in streaming aggregates, mounds and migrating slugs. Using a highly sensitive cAMP-FRET reporter, we have now been able to measure periodically propagating cAMP waves directly in these multicellular structures. In slugs cAMP waves are periodically initiated in the tip and propagate backward through the prespore zone. Altered cAMP signalling dynamics in mutants with developmental defects strongly support a key functional role for cAMP waves in multicellular Dictyostelium morphogenesis. These findings thus show that propagating cAMP not only control the initial aggregation process but continue to be the long range cell-cell communication mechanism guiding cell movement during multicellular Dictyostelium morphogenesis at the mound and slugs stages.

Cyclic di-AMP-mediated interaction between Mycobacterium tuberculosis ΔcnpB and macrophages implicates a novel strategy for improving BCG vaccination.

Cyclic di-AMP (c-di-AMP) has been shown to play an important role in bacterial physiology and pathogen-host interactions. We previously reported that deletion of the sole c-di-AMP phosphodiesterase-encoding gene (cnpB) in Mycobacterium tuberculosis (Mtb) led to significant virulence attenuation. In this study, we found that ΔcnpB of M. bovisbacillus Calmette-Guerin (BCG) was unable to secrete c-di-AMP, which differs from Mtb ΔcnpB. We infected bone marrow-derived macrophages (BMDMs) with c-di-AMP-associated mutants generated from both Mtb and BCG. Our results showed that upon infection with Mtb ΔcnpB, BMDMs of wildtype mice secreted a large amount of interferon-ß (IFN-ß) post-infection similarly as we reported previously. In contrast, the response was less pronounced with BMDMs isolated from cGAS-/- mice and was nearly abolished with BMDMs prepared from STING-/- mice. Deletion of the region of difference 1 (RD1) locus in Mtb ΔcnpB did not alter the c-di-AMP secretion of ΔcnpB but eliminated the IFN-ß production in the infected cells. In contrast, neither BCG ΔcnpB nor a recombinant BCG ΔcnpB with a pRD1 cosmid induced a type I interferon response. Interestingly, multiple studies have demonstrated that type I IFN enhances BCG's immunity. Thus, amending BCG based on our findings might improve BCG vaccination.

The Dunce cAMP phosphodiesterase PDE-4 negatively regulates G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the Caenorhabditis elegans synaptic signaling network.

Forward genetic screens for mutations that rescue the paralysis of ric-8 (Synembryn) reduction-of-function mutations frequently reveal mutations that cause hyperactivation of one or more components of the G alpha(s) pathway. Here, we report that one of these mutations strongly reduces the function of the Dunce cAMP phosphodiesterase PDE-4 by disrupting a conserved active site residue. Loss of function and neural overexpression of PDE-4 have profound and opposite effects on locomotion rate, but drug-response assays suggest that loss of PDE-4 function does not affect steady-state acetylcholine release or reception. Our genetic analysis suggests that PDE-4 regulates both G alpha(s)-dependent and G alpha(s)-independent cAMP pools in the neurons controlling locomotion rate. By immunostaining, PDE-4 is strongly expressed throughout the nervous system, where it localizes to small regions at the outside boundaries of synaptic vesicle clusters as well as intersynaptic regions. The synaptic subregions containing PDE-4 are distinct from those containing active zones, as indicated by costaining with an antibody against the long form of UNC-13. This highly focal subsynaptic localization suggests that PDE-4 may exert its effects by spatially regulating intrasynaptic cAMP pools.

Postsynaptic protein kinase A reduces neuronal excitability in response to increased synaptic excitation in the Drosophila CNS.

Previous work has identified a role for synaptic activity in the development of excitable properties of motoneurons in the Drosophila embryo. In this study the underlying mechanism that enables two such neurons, termed aCC and RP2, to respond to increased exposure to synaptic excitation is characterized. Synaptic excitation is increased in genetic backgrounds that lack either a cAMP-specific phosphodiesterase (EC:3.1.4, dunce) or acetylcholinesterase (EC:3.1.1.7, ace), the enzyme that terminates the endogenous cholinergic excitation of these motoneurons. Analysis of membrane excitability in aCC/RP2, in either background, shows that these neurons have a significantly reduced capability to fire action potentials (APs) in response to injection of depolarizing current. Analysis of underlying voltage-gated currents show that this effect is associated with a marked reduction in magnitude of the voltage-dependent inward Na+ current (INa). Partially blocking INa in these motoneurons, using low concentrations of TTX, demonstrates that a reduction of INa is, by itself, sufficient to reduce membrane excitability. An analysis of firing implicates an increased AP threshold to underlie the reduction in membrane excitability observed because of heightened exposure to synaptic excitation. Genetic or pharmacological manipulations that either elevate cAMP or increase protein kinase A (PKA) activity in wild-type aCC/RP2 mimic both the reductions in membrane excitability and INa. In comparison, increasing cAMP catabolism or inhibition of PKA activity is sufficient to block the induction of these activity-dependent changes. The induced changes in excitability can be rapid, occurring within 5 min of exposure to a membrane-permeable cAMP analog, indicative that threshold can be regulated in these neurons by a post-translational mechanism that is dependent on phosphorylation.

Phosphodiesterase 8B and cyclic AMP signaling in the adrenal cortex.

Bilateral adrenocortical hyperplasia (BAH) in humans and mice has been recently linked to phosphodiesterase (PDE) 8B (PDE8B) and 11 (PDE11A) defects. These findings have followed the discovery that defects of primary genes of the cyclic monophosphatase (cAMP) signaling pathway, such as guanine nucleotide binding alpha subunit and PRKAR1A, are involved in the pathogenesis of BAH in humans; complete absence of Prkar1a in the adrenal cortex of mice also led to pathology that mimicked the human disease. Here, we review the most recent findings in human and mouse studies on PDE8B, a cAMP-specific PDE that appears to be highly expressed in the adrenal cortex and whose deficiency may underlie predisposition to BAH and possibly other human diseases.

Antidepressant-like profile and reduced sensitivity to rolipram in mice deficient in the PDE4D phosphodiesterase enzyme.

Pharmacological inhibition of type 4 cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE4) produces antidepressant-like effects in animals; however, it is not known which of the four PDE4 subtypes mediates these actions. In the present study, immunoblot analysis showed loss of phosphodiesterase 4D (PDE4D) expression in the cerebral cortex and hippocampus of PDE4D knockout (PDE4D-/-) mice, but unchanged PDE4A and PDE4B expression, relative to the wild type (PDE4D+/+) and heterozygous knockout (PDE4D+/-) mice. This reduced expression was accompanied by a reduction in PDE4 activity, while non-PDE4 activity was unchanged. PDE4D-/- mice exhibited decreased immobility in tail-suspension and forced-swim tests, which is indicative of an antidepressant-like effect on behavior. Desipramine and fluoxetine produced similar antidepressant-like effects in all three genotypes, even though their behavioral baselines differed markedly. By contrast, the PDE4 inhibitor rolipram only produced antidepressant-like effects in PDE4D+/+ mice. Consistent with this, rolipram potentiated isoproterenol-induced cyclic AMP formation only in the PDE4D+/+ mice. These results suggest that PDE4D is an essential mediator of the antidepressant-like effects of rolipram, and that PDE4D-regulated cyclic adenosine monophosphate signaling may play a role in the pathophysiology and pharmacotherapy of depression.

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

BACKGROUND: HIV-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. RESULTS: PDE8A 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. CONCLUSIONS: PDE8A 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.

Modulation of Leydig cell function by cyclic nucleotide phosphodiesterase 8A.

Leydig cells produce testosterone in the testes under the pulsatile control of pituitary luteinizing hormone (LH). cAMP is the intracellular messenger for LH action on steroidogenesis, and pharmacological evidence indicates that the response to LH can be modulated by cyclic nucleotide phosphodiesterases (PDEs). However the types and roles of the PDEs present in Leydig cells have not been fully defined. We report here that PDE8A is expressed in Leydig cells, and using PDE8A knockout mice we provide evidence that PDE8A is a key regulator of LH signaling and steroidogenesis. A 4-fold increase in the sensitivity to LH for testosterone production was detected in Leydig cells isolated from PDE8A knockout mice. In Leydig cells from wild-type mice, 3-isobutyl-1-methylxanthine, a compound that inhibits all cAMP PDEs except PDE8A, elicited only a small increase in the sensitivity of testosterone production to LH. However, in the PDE8-null mice, the effect of this inhibitor is much more pronounced. These observations indicate that PDE8A and at least one other PDE control the same or a complementary pool of cAMP that mediates LH-regulated steroidogenesis. Overall, these results suggest that pharmacological manipulation of PDE8A, alone or in combination with other PDEs present in Leydig cells, may be exploited to modulate testosterone synthesis and possibly to treat various conditions where the local levels of this androgen need to be altered.

Protein kinase B/Akt phosphorylation of PDE3A and its role in mammalian oocyte maturation.

cGMP-inhibited cAMP phosphodiesterase 3A (PDE3A) is expressed in mouse oocytes, and its function is indispensable for meiotic maturation as demonstrated by genetic ablation. Moreover, PDE3 activity is required for insulin/insulin-like growth factor-1 stimulation of Xenopus oocyte meiotic resumption. Here, we investigated the cAMP-dependent protein kinase B (PKB)/Akt regulation of PDE3A and its impact on oocyte maturation. Cell-free incubation of recombinant mouse PDE3A with PKB/Akt or cAMP-dependent protein kinase A catalytic subunits leads to phosphorylation of the PDE3A protein. Coexpression of PDE3A with constitutively activated PKB/Akt (Myr-Akt) increases PDE activity as well as its phosphorylation state. Injection of pde3a mRNA potentiates insulin-dependent maturation of Xenopus oocytes and rescues the phenotype of pde3(-/-) mouse oocytes. This effect is greatly decreased by mutation of any of the PDE3A serines 290-292 to alanine in both Xenopus and mouse. Microinjection of myr-Akt in mouse oocytes causes in vitro meiotic maturation and this effect requires PDE3A. Collectively, these data indicate that activation of PDE3A by PKB/Akt-mediated phosphorylation plays a role in the control of PDE3A activity in mammalian oocytes.

Phosphodiesterase 4D deficiency in the ryanodine-receptor complex promotes heart failure and arrhythmias.

Phosphodiesterases (PDEs) regulate the local concentration of 3',5' cyclic adenosine monophosphate (cAMP) within cells. cAMP activates the cAMP-dependent protein kinase (PKA). In patients, PDE inhibitors have been linked to heart failure and cardiac arrhythmias, although the mechanisms are not understood. We show that PDE4D gene inactivation in mice results in a progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias. The phosphodiesterase 4D3 (PDE4D3) was found in the cardiac ryanodine receptor (RyR2)/calcium-release-channel complex (required for excitation-contraction [EC] coupling in heart muscle). PDE4D3 levels in the RyR2 complex were reduced in failing human hearts, contributing to PKA-hyperphosphorylated, "leaky" RyR2 channels that promote cardiac dysfunction and arrhythmias. Cardiac arrhythmias and dysfunction associated with PDE4 inhibition or deficiency were suppressed in mice harboring RyR2 that cannot be PKA phosphorylated. These data suggest that reduced PDE4D activity causes defective RyR2-channel function associated with heart failure and arrhythmias.

Specific role of phosphodiesterase 4B in lipopolysaccharide-induced signaling in mouse macrophages.

Cyclic nucleotide signaling functions as a negative modulator of inflammatory cell responses, and type 4 phosphodiesterases (PDE4) are important regulators of this pathway. In this study, we provide evidence that only one of the three PDE4 genes expressed in mouse peritoneal macrophages is involved in the control of TLR signaling. In these cells, LPS stimulation of TLR caused a major up-regulation of PDE4B but not the paralogs PDE4A or PDE4D. Only ablation of PDE4B impacted LPS signaling and TNF-alpha production. TNF-alpha mRNA and protein were decreased by >50% in PDE4B-/-, but not in PDE4A-/- or PDE4D-/- macrophages. The PDE4 selective inhibitors rolipram and roflumilast had no additional inhibitory effect in macrophages deficient in PDE4B, but suppressed the TNF-alpha response in the other PDE4 null cells. The inhibition of TNF-alpha production that follows either genetic ablation or acute inhibition of PDE4B is cAMP-dependent and requires protein kinase A activity. However, no global changes in cAMP concentration were observed in the PDE4B-/- macrophages. Moreover, ablation of PDE4B protected mice from LPS-induced shock, suggesting that altered TLR signaling is retained in vivo. These findings demonstrate the highly specialized function of PDE4B in macrophages and its critical role in LPS signaling. Moreover, they provide proof of concept that a PDE4 inhibitor with subtype selectivity retains useful pharmacological effects.

Mutants of Serratia marcescens lacking cyclic nucleotide phosphodiesterase activity and requiring cyclic 3',5'-AMP for the utilization of various carbohydrates.

Adenine requiring mutants of Serratia marcescens SM-6-F'lac+ have been found to grow well in minimal-glucose medium solely supplemented with cAMP. From one of these ade strains double mutants (called ade cpd) were isolated which could no longer utilize cAMP but which still grew on 5'AMP. Dialyzed cell extracts (soluble fraction) of the double mutants, assayed for cAMP phosphodiesterase, were unable to hydrolyze cAMP whereas cell extracts of the parental strains yielded 5'AMP at a rate of 1.6-2.0 mumoles min-1 mg-1 protein. The loss of the phosphodiesterase activity in S. marcescens cpd W 1181 did not cause an accumulation of large amounts of cAMP as was found for the diesterase-negative mutant AB257pc-1 of Escherichia coli. The induced synthesis of beta-galactosidase in mutant cpd W 1181 showed about the same sensitivity to transient and permanent catabolite (glucose) repression as the corresponding cpd+ strain. Starting from S. marcescens cpd W 1182 three independent double mutants (called cpd cya) were isolated which required exogenous cAMP for utilizing various carbohydrates as carbon source, for motility and for the formation of extracellular lipase and the red pigment prodigiosine. The intracellular concentration of cAMP in these mutants, grown in nutrient broth, was 40-60% of that of the parental strain which is about 4 x 10(-4) M. However, the adenylate cyclase in cell extracts of the mutants W 1237 and W 1270 was like that of the corresponding cya+ strain (about 2 x 10(-2) mumoles min-1 mg-1 protein).

Absence of muscarinic cholinergic airway responses in mice deficient in the cyclic nucleotide phosphodiesterase PDE4D.

Muscarinic cholinergic signaling plays an essential role in the control of the normal airway functions and in the development of pulmonary pathologies including asthma. In this paper we demonstrate that the airways of mice deficient in a cAMP-specific phosphodiesterase (PDE4D) are no longer responsive to cholinergic stimulation. Airway hyperreactivity that follows exposure to antigen was also abolished in PDE4D(-/-) mice, despite an apparently normal lung inflammatory infiltration. The loss of cholinergic responsiveness was specific to the airway, not observed in the heart, and was associated with a loss of signaling through muscarinic receptors with an inability to decrease cAMP accumulation. These findings demonstrate that the PDE4D gene plays an essential role in cAMP homeostasis and cholinergic stimulation of the airway, and in the development of hyperreactivity. In view of the therapeutic potentials of PDE4 inhibitors, our findings provide the rationale for novel strategies that target a single PDE isoenzyme.

Phosphodiesterase 7A-deficient mice have functional T cells.

Phosphodiesterases (PDEs) are enzymes which hydrolyze the cyclic nucleotide second messengers, cAMP and cGMP. In leukocytes, PDEs are responsible for depletion of cAMP which broadly suppresses cell functions and cellular responses to many activation stimuli. PDE7A has been proposed to be essential for T lymphocyte activation based on its induction during cell activation and the suppression of proliferation and IL-2 production observed following inhibition of PDE7A expression using a PDE7A antisense oligonucleotide. These observations have led to the suggestion that selective PDE7 inhibitors could be useful in the treatment of T cell-mediated autoimmune diseases. In the present report, we have used targeted gene disruption to examine the role PDE7A plays in T cell activation. In our studies, PDE7A knockout mice (PDE7A(-/-)) showed no deficiencies in T cell proliferation or Th1- and Th2-cytokine production driven by CD3 and CD28 costimulation. Unexpectedly, the Ab response to the T cell-dependent Ag, keyhole limpet hemocyanin, in the PDE7A(-/-) mice was found to be significantly elevated. The results from our studies strongly support the notion that PDE7A is not essential for T cell activation.