Phosphodiesterase isoform‐specific expression induced by traumatic brain injury

Traumatic brain injury (TBI) results in significant inflammation which contributes to the evolving pathology. Previously, we have demonstrated that cyclic AMP (cAMP), a molecule involved in inflammation, is down‐regulated after TBI. To determine the mechanism by which cAMP is down‐regulated after TBI, we determined whether TBI induces changes in phosphodiesterase (PDE) expression. Adult male Sprague Dawley rats received moderate parasagittal fluid‐percussion brain injury (FPI) or sham injury, and the ipsilateral, parietal cortex was analyzed by western blotting. In the ipsilateral parietal cortex, expression of PDE1A, PDE4B2, and PDE4D2, significantly increased from 30 min to 24 h post‐injury. PDE10A significantly increased at 6 and 24 h after TBI. Phosphorylation of PDE4A significantly increased from 6 h to 7 days post‐injury. In contrast, PDE1B, PD4A5, and PDE4A8 significantly decreased after TBI. No changes were observed with PDE1C, PDE3A, PDE4B1/3, PDE4B4, PDE4D3, PDE4D4, PDE8A, or PDE8B. Co‐localization studies showed that PDE1A, PDE4B2, and phospho‐PDE4A were neuronally expressed, whereas PDE4D2 was expressed in neither neurons nor glia. These findings suggest that therapies to reduce inflammation after TBI could be facilitated with targeted therapies, in particular for PDE1A, PDE4B2, PDE4D2, or PDE10A.     

Differential expression and function of phosphodiesterase 4 (PDE4) subtypes in human primary CD4+ T cells: predominant role of PDE4D

Type 4 phosphodiesterases (PDE4) are critical regulators in TCR signaling by attenuating the negative constraint of cAMP. In this study, we show that anti-CD3/CD28 stimulation of human primary CD4(+) T cells increases the expression of the PDE4 subtypes PDE4A, PDE4B, and PDE4D in a specific and time-dependent manner. PDE4A and PDE4D mRNAs as well as enzyme activities were up-regulated within 5 days, PDE4B showed a transient up-regulation with highest levels after 24 h. The induction was shown to be independent of different stimulation conditions and was similar in naive and memory T cell subpopulations. To elucidate the functional impact of individual PDE4 subtypes on T cell function, we used PDE4 subtype-specific short-interfering RNAs (siRNAs). Knockdown of either PDE4B or PDE4D inhibited IL-2 release 24 h after stimulation (time point of maximal IL-2 concentrations) to an extent similar to that observed with the panPDE4 inhibitor RP73401 (piclamilast). Substantial amounts of IFN-gamma or IL-5 were measured only at later time points. siRNA targeting PDE4D showed a predominant inhibitory effect on these cytokines measured after 72 h. However, the inhibition of all cytokines was most effective when PDE4 siRNAs were applied in combination. Although the effect of PDE4 inhibition on T cell proliferation is small, the PDE4D-targeting siRNA alone was as effective as the panPDE4 inhibitor, whereas PDE4A or PDE4B siRNAs had hardly an effect. In summary, individual PDE4 subtypes have overall nonredundant, but complementary, time-dependent roles in propagating various T cell functions and PDE4D is the form likely playing a predominant role.     

Human PDE4A8, a novel brain-expressed PDE4 cAMP-specific phosphodiesterase that has undergone rapid evolutionary change

We have isolated cDNAs encoding PDE4A8 (phosphodiesterase 4 isoform A8), a new human cAMP-specific PDE4 isoform encoded by the PDE4A gene. PDE4A8 has a novel N-terminal region of 85 amino acids that differs from those of the related 'long' PDE4A4, PDE4A10 and PDE4A11 isoforms. The human PDE4A8 N-terminal region has diverged substantially from the corresponding isoforms in the rat and other mammals, consistent with rapid evolutionary change in this region of the protein. When expressed in COS-7 cells, PDE4A8 localized predominantly in the cytosol, but approx. 20% of the enzyme was associated with membrane fractions. Cytosolic PDE4A8 was exquisitely sensitive to inhibition by the prototypical PDE4 inhibitor rolipram (IC(50) of 11+/-1 nM compared with 1600 nM for PDE4A4), but was less sensitive to inhibition by cilomilast (IC(50) of 101+/-7 nM compared with 61 nM for PDE4A4). PDE4A8 mRNA was found to be expressed predominantly in skeletal muscle and brain, a pattern that differs from the tissue expression of other human PDE4 isoforms and also from that of rat PDE4A8. Immunohistochemical analysis showed that PDE4A8 could be detected in discrete regions of human brain, including the cerebellum, spinal cord and cerebral cortex. The unique tissue distribution of PDE4A8, combined with the evolutionary divergence of its N-terminus, suggest that this isoform may have a specific function in regulating cAMP levels in human skeletal muscle and brain.     

The unique N-terminal domain of the cAMP phosphodiesterase PDE4D4 allows for interaction with specific SH3 domains

Of the five PDE4D isoenzymes, only the PDE4D4 cAMP specific phosphodiesterase was able to bind to SH3 domains. Only PDE4D4 and PDE4A5, but not any other PDE4A, B, C and D isoforms expressed in rat brain, bound to src, lyn and fyn kinase SH3 domains. Purified PDE4D4 could bind to purified lyn SH3. PDE4D4 and PDE4A5 both exhibited selectivity for binding the SH3 domains of certain proteins. PDE4D4 did not bind to WW domains. We suggest that an important function of the unique N-terminal region of PDE4D4 may be to allow for association with certain SH3 domain-containing proteins.     

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.     

Occupancy of the catalytic site of the PDE4A4 cyclic AMP phosphodiesterase by rolipram triggers the dynamic redistribution of this specific isoform in living cells through a cyclic AMP independent process

In cells transfected to express wild-type PDE4A4 cAMP phosphodiesterase (PDE), the PDE4 selective inhibitor rolipram caused PDE4A4 to relocalise so as to form accretion foci. This process was followed in detail in living cells using a PDE4A4 chimera formed with Green Fluorescent Protein (GFP). The same pattern of behaviour was also seen in chimeras of PDE4A4 formed with various proteins and peptides, including LimK, RhoC, FRB and the V5-6His tag. Maximal PDE4A4 foci formation, occurred over a period of about 10 h, was dose-dependent on rolipram and was reversible upon washout of rolipram. Inhibition of protein synthesis, using cycloheximide, but not PKA activity with H89, inhibited foci generation. Foci formation was elicited by Ro20-1724 and RS25344 but not by either Ariflo or RP73401, showing that not all PDE4 selective inhibitors had this effect. Ariflo and RP73401 dose-dependently antagonised rolipram-induced foci formation and dispersed rolipram pre-formed foci as did the adenylyl cyclase activator, forskolin. Foci formation showed specificity for PDE4A4 and its rodent homologue, PDE4A5, as it was not triggered in living cells expressing the PDE4B2, PDE4C2, PDE4D3 and PDE4D5 isoforms as GFP chimeras. Altered foci formation was seen in the Deltab-LR2-PDE4A4 construct, which deleted a region within LRZ, showing that appropriate linkage between the N-terminal portion of PDE4A4 and the catalytic unit of PDE4A4 was needed for foci formation. Certain single point mutations within the PDE4A4 catalytic site (His505Asn, His506Asn and Val475Asp) were shown to ablate foci formation but still allow rolipram inhibition of PDE4A4 catalytic activity. We suggest that the binding of certain, but not all, PDE4 selective inhibitors to PDE4A4 induces a conformational change in this isoform by 'inside-out' signalling that causes it to redistribute in the cell. Displacing foci-forming inhibitors with either cAMP or inhibitors that do not form foci can antagonise this effect. Specificity of this effect for PDE4A4 and its homologue PDE4A5 suggests that interplay between the catalytic site and the unique N-terminal region of these isoforms is required. Thus, certain PDE4 selective inhibitors may exert effects on PDE4A4 that extend beyond simple catalytic inhibition. These require protein synthesis and may lead to redistribution of PDE4A4 and any associated proteins. Foci formation of PDE4A4 may be of use in probing for conformational changes in this isoform and for sub-categorising PDE4 selective inhibitors.     

mRNA expression and antilipolytic role of phosphodiesterase 4 in rat adipocytes in vitro

Adipocyte lipolysis is dependent on an increase in the intracellular concentration of cAMP. Intracellular phosphodiesterases (PDEs) hydrolyze cAMP and limit stimulation of lipolysis. In the present study, the mRNA expression of PDE4 subtypes and the antilipolytic role of PDE4 in rat adipocytes were investigated. Fragments encoding PDE4A (233 bp), PDE4B (786 bp), PDE4C (539 bp), and PDE4D (262 bp) sequences were amplified by RT-PCR. The mRNA expression of PDE4 subtypes (A, B, C, D) determined by real-time quantitative PCR was 7, 18.7, 18.9, and 7.2% relative to PDE3B. Inhibition of PDE4 by rolipram increased basal lipolysis and reversed in part prostaglandin E2 antilipolysis. The combination of PDE3 and PDE4 inhibitors synergistically reversed both prostaglandin E2 and phenylisopropyl adenosine antilipolysis. Stimulation of adipocytes with prostaglandin E2 increased total PDE activity and PDE3 activity measured by hydrolysis of 3[H]cAMP by the particulate fraction of adipocytes. The present study confirmed that mRNAs for all four PDE4 subtypes were expressed in rat adipocytes, with PDE4B and PDE4C predominant. Moreover, PDE4 not only limits the rate of basal lipolysis but also contributes to prostaglandin E2 antilipolysis in rat adipocytes.     

In resting COS1 cells a dominant negative approach shows that specific, anchored PDE4 cAMP phosphodiesterase isoforms gate the activation, by basal cyclic AMP production, of AKAP-tethered protein kinase A type II located in the centrosomal region

We employ a novel, dominant negative approach to identify a key role for certain tethered cyclic AMP specific phosphodiesterase-4 (PDE4) isoforms in regulating cyclic AMP dependent protein kinase A (PKA) sub-populations in resting COS1 cells. A fraction of PKA is clearly active in resting COS1 cells and this activity increases when cells are treated with the selective PDE4 inhibitor, rolipram. Point mutation of a critical, conserved aspartate residue in the catalytic site of long PDE4A4, PDE4B1, PDE4C2 and PDE4D3 isoforms renders them catalytically inactive. Overexpressed in resting COS1 cells, catalytically inactive forms of PDE4C2 and PDE4D3, but not PDE4A4 and PDE4B1, are constitutively PKA phosphorylated while overexpressed active versions of all these isoforms are not. Inactive and active versions of all these isoforms are PKA phosphorylated in cells where protein kinase A is maximally activated with forskolin and IBMX. By contrast, rolipram challenge of COS1 cells selectively triggers the PKA phosphorylation of recombinant, active PDE4D3 and PDE4C2 but not recombinant, active PDE4A4 and PDE4B1. Purified, recombinant PDE4D3 and PDE4A4 show a similar dose-dependency for in vitro phosphorylation by PKA. Disruption of the tethering of PKA type-II to PKA anchor proteins (AKAPs), achieved using the peptide Ht31, prevents inactive forms of PDE4C2 and PDE4D3 being constitutively PKA phosphorylated in resting cells as does siRNA-mediated knockdown of PKA-RII, but not PKA-RI. PDE4C2 and PDE4D3 co-immunoprecipitate from COS1 cell lysates with 250 kDa and 450 kDa AKAPs that tether PKA type-II and not PKA type-I. PKA type-II co-localises with AKAP450 in the centrosomal region of COS1 cells. The perinuclear distribution of recombinant, inactive PDE4D3, but not inactive PDE4A4, overlaps with AKAP450 and PKA type-II. The distribution of PKA phosphorylated inactive PDE4D3 also overlaps with that of AKAP450 in the centrosomal region of COS1 cells. We propose that a novel role for PDE4D3 and PDE4C2 is to gate the activation of AKAP450-tethered PKA type-II localised in the perinuclear region under conditions of basal cAMP generation in resting cells.     

Development of a fission yeast-based high-throughput screen to identify chemical regulators of cAMP phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3',5'-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens.     

Structures of the four subfamilies of phosphodiesterase-4 provide insight into the selectivity of their inhibitors

PDE4 (phosphodiesterase-4)-selective inhibitors have attracted much attention as potential therapeutics for the treatment of both depression and major inflammatory diseases, but their practical application has been compromised by side effects. A possible cause for the side effects is that current PDE4-selective inhibitors similarly inhibit isoforms from all four PDE4 subfamilies. The development of PDE4 subfamily-selective inhibitors has been hampered by a lack of structural information. In the present study, we rectify this by providing the crystal structures of the catalytic domains of PDE4A, PDE4B and PDE4D in complex with the PDE4 inhibitor NVP {4-[8-(3-nitrophenyl)-[1,7]naphthyridin-6-yl]benzoic acid} as well as the unliganded PDE4C structure. NVP binds in the same conformation to the deep cAMP substrate pocket and interacts with the same residues in each instance. However, detailed structural comparison reveals significant conformational differences. Although the active sites of PDE4B and PDE4D are mostly comparable, PDE4A shows significant displacements of the residues next to the invariant glutamine residue that is critical for substrate and inhibitor binding. PDE4C appears to be more distal from other PDE4 subfamilies, with certain key residues being disordered. Our analyses provide the first structural basis for the development of PDE4 subfamily-selective inhibitors.     

Critical role of PDE4D in beta2-adrenoceptor-dependent cAMP signaling in mouse embryonic fibroblasts

One of the defining properties of beta2-adrenergic receptor (beta(2)AR) signaling is the transient and rapidly reversed accumulation of cAMP. Here we have investigated the contribution of different PDE4 proteins to the generation of this transient response. To this aim, mouse embryonic fibroblasts deficient in PDE4A, PDE4B, or PDE4D were generated, and the regulation of PDE activity, the accumulation of cAMP, and CREB phosphorylation in response to isoproterenol were monitored. Ablation of PDE4D, but not PDE4A or PDE4B, had a major effect on the beta-agonist-induced PDE activation, with only a minimal increase in PDE activity being retained in PDE4D knock-out (KO) cells. Accumulation of cAMP was markedly enhanced, and the kinetics of cAMP accumulation were altered in their properties in PDE4DKO but not PDE4BKO cells. Modest effects were observed in PDE4AKO mouse embryonic fibroblasts. The return to basal levels of both cAMP accumulation and CREB phosphorylation was greatly delayed in the PDE4DKO cells, suggesting that PDE4D is critical for dissipation of the beta2AR stimulus. This effect of PDE4D ablation was in large part due to inactivation of a negative feedback mechanism consisting of the PKA-mediated activation of PDE4D in response to elevated cAMP levels, as indicated by experiments using the cAMP-dependent protein kinase inhibitors H89 and PKI. Finally, PDE4D ablation affected the kinetics of beta2AR desensitization as well as the interaction of the receptor with Galphai. These findings demonstrate that PDE4D plays a major role in shaping the beta2AR signal.     

Cross talk between NO and cyclic nucleotide phosphodiesterases in the modulation of signal transduction in blood vessel.

An increase in cAMP and/or cGMP induces vasodilation which could be potentiated by endothelium or NO-donors. Cyclic nucleotide phosphodiesterases (PDE) are differently distributed in vascular tissues. cAMP hydrolyzing PDE isozymes in endothelial cells are represented by PDE2 (cGMP stimulated-PDE) and PDE4 (cGMP insensitive-PDE), whereas in smooth muscle cells PDE3 (cGMP inhibited-PDE) and PDE4 are present. To investigate the role of NO in vasodilation induced by PDE inhibitors, we studied the effects of PDE3- or PDE4-inhibitor alone and their combination on cyclic nucleotide levels, on relaxation of precontracted aorta and on protein kinase implication. Furthermore, the direct effect of dinitrosyl iron complex (DNIC) was studied on purified recombinant PDE4B. The results show that: 1) in endothelial cells PDE4 inhibition may up-regulate basal production of NO, this effect being potentiated by PDE2 inhibition; 2) in smooth muscle cGMP produced by NO inhibits PDE3 and increases cAMP level allowing PDE4 to participate in vascular contraction; 3) protein kinase G mediates the relaxing effects of PDE3 or PDE4 inhibition. 4) DNIC inhibits non competitively PDE4B indicating a direct effect of NO on PDE4 which could explain an additive vasodilatory effect of NO. A direct and a cGMP related cross-talk between NO and cAMP-PDEs, may participate into the vasomodulation mediated by cAMP activation of protein kinase G.     

Discovery of potent,selective, bioavailable phosphodiesterase 2 (PDE2) inhibitors active in an osteoarthritis pain model,Part I: Transformation of selective pyrazolodiazepinone phosphodiesterase 4 (PDE4) inhibitors into selective PDE2 inhibitors

We identified potent, selective PDE2 inhibitors by optimizing residual PDE2 activity in a series of PDE4 inhibitors, while simultaneously minimizing PDE4 activity. These newly designed PDE2 inhibitors bind to the PDE2 enzyme in a cGMP-like mode in contrast to the cAMP-like binding mode found in PDE4. Structure activity relationship studies coupled with an inhibitor bound crystal structure in the active site of the catalytic domain of PDE2 identified structural features required to minimize PDE4 inhibition while simultaneously maximizing PDE2 inhibition.