Biomedical subjects

Miles D Houslay

Publications and source records attributed to Miles D Houslay.

At least 19 recordsLinked to original sources

PubMed2006

Compartmentalization of cAMP-dependent signaling by phosphodiesterase-4D is involved in the regulation of vasopressin-mediated water reabsorption in renal principal cells.

The cAMP/protein kinase A (PKA)-dependent insertion of water channel aquaporin-2 (AQP2)-bearing vesicles into the plasma membrane in renal collecting duct principal cells (AQP2 shuttle) constitutes the molecular basis of arginine vasopressin (AVP)-regulated water reabsorption. cAMP/PKA signaling systems are compartmentalized by A kinase anchoring proteins (AKAP) that tether PKA to subcellular sites and by phosphodiesterases (PDE) that terminate PKA signaling through hydrolysis of localized cAMP. In primary cultured principal cells, AVP causes focal activation of PKA. PKA and cAMP-specific phosphodiesterase-4D (PDE4D) are located on AQP2-bearing vesicles. The selective PDE4 inhibitor rolipram increases AKAP-tethered PKA activity on AQP2-bearing vesicles and enhances the AQP2 shuttle and thereby the osmotic water permeability. AKAP18delta, which is located on AQP2-bearing vesicles, directly interacts with PDE4D and PKA. In response to AVP, PDE4D and AQP2 translocate to the plasma membrane. Here PDE4D is activated through PKA phosphorylation and reduces the osmotic water permeability. Taken together, a novel, compartmentalized, and physiologically relevant cAMP-dependent signal transduction module on AQP2-bearing vesicles, comprising anchored PDE4D, AKAP18delta, and PKA, has been identified.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

PGE(1) stimulation of HEK293 cells generates multiple contiguous domains with different [cAMP]: role of compartmentalized phosphodiesterases.

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Helix-1 of the cAMP-specific phosphodiesterase PDE4A1 regulates its phospholipase-D-dependent redistribution in response to release of Ca2+.

The unique N-terminal regions of PDE4 cAMP-specific phosphodiesterases confer interaction with distinct signalling and scaffolding proteins. The PDE4A1 isoform is unique in being entirely membrane associated. Its N-terminal region is formed from two helices separated by a mobile hinge, where helix-2 contains a TAPAS1 domain that inserts into the lipid bilayer in a Ca2+-triggered fashion. Here we show that helix-1 is important for intracellular targeting of PDE4A1 in living cells, facilitating membrane association, targeting to the trans-Golgi stack and conferring Ca2+-stimulated intracellular redistribution in a manner that is dependent on the phospholipase-D-mediated generation of phosphatidic acid. The LxDFF motif within helix-1 is pivotal to this, where Leu4-Phe6-Phe7 forms a compact hydrophobic pocket on one side of helix-1 whereas Asp5, located on the opposite face of helix-1, provides the Ca2+-regulation site. Mutation of Asp5 to Ala or the release of Ca2+ from intracellular stores de-restricts trans-Golgi localisation of PDE4A1 allowing it to redistribute in cells in a phosphatidic-acid-dependent manner. This study provides the first evidence for Ca2+-triggered relocalisation of a cAMP phosphodiesterase and indicates a potential means for allowing cross-talk between the cAMP, phospholipase D and Ca2+-signalling pathways.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

A RSK(y) relationship with promiscuous PKA.

Where, when, and with "whom" do molecules interact? Such relations in space and time are key concepts that currently engage investigators of cellular signaling processes. The notion of compartmentalized signaling grew out of studies of adenosine 3',5'-monophosphate (cAMP) signaling processes, and this area continues to generate exciting new paradigms. Distinct clouds of cAMP are formed and shaped within cells by tethered cAMP phosphodiesterases (PDEs). AKAPosomes, formed from distinct subpopulations of cAMP-dependent protein kinase (PKA) tethered to anchoring proteins (AKAPs) together with specific substrate molecules, interpret these gradients to generate individualized responses. PKA activity is also regulated by the interaction of other proteins with the regulatory (R) or catalytic (C) subunits of PKA, and a mechanism has been uncovered in which ribosomal S6 kinase (RSK1) interacts with either PKA subunit, depending on whether RSK1 has been phosphorylated and activated by extracellular signal-regulated kinase (ERK). Thus, inactive RSK1 binds the RI subunit of PKA to sensitize it to activation, whereas activated RSK1 binds the C subunit to desensitize PKA to cAMP activation. Cross-talk between the key cAMP and ERK signaling pathways provides a mechanism that, along with distinct mechanisms of both positive and negative attenuation provided by Raf and PDE4 isoforms, can be tailored on a cell type-specific basis.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Scanning peptide array analyses identify overlapping binding sites for the signalling scaffold proteins, beta-arrestin and RACK1, in cAMP-specific phosphodiesterase PDE4D5.

The cAMP-specific phosphodiesterase PDE4D5 can interact with the signalling scaffold proteins RACK (receptors for activated C-kinase) 1 and beta-arrestin. Two-hybrid and co-immunoprecipitation analyses showed that RACK1 and beta-arrestin interact with PDE4D5 in a mutually exclusive manner. Overlay studies with PDE4D5 scanning peptide array libraries showed that RACK1 and beta-arrestin interact at overlapping sites within the unique N-terminal region of PDE4D5 and at distinct sites within the conserved PDE4 catalytic domain. Screening scanning alanine substitution peptide arrays, coupled with mutagenesis and truncation studies, allowed definition of RACK1 and beta-arrestin interaction sites. Modelled on the PDE4D catalytic domain, these form distinct well-defined surface-exposed patches on helices-15-16, for RACK1, and helix-17 for beta-arrestin. siRNA (small interfering RNA)-mediated knockdown of RACK1 in HEK-293 (human embryonic kidney) B2 cells increased beta-arrestin-scaffolded PDE4D5 approx. 5-fold, increased PDE4D5 recruited to the beta2AR (beta2-adrenergic receptor) upon isoproterenol challenge approx. 4-fold and severely attenuated (approx. 4-5 fold) both isoproterenol-stimulated PKA (protein kinase A) phosphorylation of the beta2AR and activation of ERK (extracellular-signal-regulated kinase). The ability of a catalytically inactive form of PDE4D5 to exert a dominant negative effect in amplifying isoproterenol-stimulated ERK activation was ablated by a mutation that blocked the interaction of PDE4D5 with beta-arrestin. In the present study, we show that the signalling scaffold proteins RACK1 and beta-arrestin compete to sequester distinct 'pools' of PDE4D5. In this fashion, alterations in the level of RACK1 expression may act to modulate signal transduction mediated by the beta2AR.

Amino Acid Sequence↗

PubMed2006

Oxidative stress employs phosphatidyl inositol 3-kinase and ERK signalling pathways to activate cAMP phosphodiesterase-4D3 (PDE4D3) through multi-site phosphorylation at Ser239 and Ser579.

RAW macrophages, which express the PDE4D3 and PDE4D5 cAMP phosphodiesterase isoforms, exhibited increased PDE4 activity when challenged with H2O2 in a fashion that was negated by treatment with the cell permeant antioxidant, N-acetyl cysteine and by diphenyleneiodonium chloride, an inhibitor of NADPH oxidase. In Cos1 cells transfected to express PDE4D3, challenge with H2O2 caused a rapid increase in both the activity and phosphorylation of PDE4D3. Lysates from H2O2-treated COS cells caused the phosphorylation of purified, recombinant PDE4D3 at two sites. One was the established ERK phosphorylation site at Ser579, located at the extreme C-terminus of the catalytic unit, and the other was a novel site at Ser239, located at the extreme N-terminus of the catalytic unit. Double Ser239Ala:Ser579Ala mutation of PDE4D3 prevented its H2O2-dependent phosphorylation both in vitro and in intact COS cells. Phosphorylation of PDE4D3 at Ser579 was ablated by treating COS cells with the MEK inhibitor, PD98059, which also negated activation. The activity of the Ser239Ala:Ser579Ala double mutant, and the Ser579Ala single PDE4D3 mutant was unaffected by H2O2 challenge of COS cells, whilst the Ser239Ala mutant was inhibited. Wortmannin inhibited the H2O2-dependent phosphorylation of PDE4D3 in COS cells by around 50%, whilst it fully ablated phosphorylation at Ser239 as well as ablating activation of PDE4D3. Neither immunodepletion of p70S6 kinase nor siRNA-mediated knockdown of mTor inhibited the H2O2-dependent phosphorylation of PDE4D3 at Ser239. Activation of PDE4D3 by challenge with H2O2 was not additive with activation through protein kinase A (PKA)-mediated phosphorylation of PDE4D3. Challenge with H2O2 did not alter PKA-mediated phosphorylation of PDE4D3 at Ser54. H2O2 dependent phosphorylation of PDE4D3, at Ser239 and Ser579, did not alter the sensitivity of PDE4D3 to inhibition by the selective PDE4 inhibitor, rolipram. An unknown protein kinase acting downstream of phosphatidyl inositol 3-kinase phosphorylates PDE4D3 at Ser239. This switches the effect of phosphorylation by ERK at Ser579 from inhibition to activation. We propose that phosphorylation at Ser239 attenuates interaction between either UCR2 or the UCR1/UCR2 module and the PDE4 catalytic unit so as to re-programme the functional outcome effect of phosphorylation by ERK. We identify a novel process through which reactive oxygen species activate long PDE4 isoforms so as to reduce cAMP levels and thereby promote inflammatory responses.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Reduced PDE4 expression and activity contributes to enhanced catecholamine-induced cAMP accumulation in adipocytes from FOXC2 transgenic mice.

Overexpression of forkhead transcription factor FOXC2 in white adipose tissue (WAT) leads to a lean phenotype resistant to diet-induced obesity. This is due, in part, to enhanced catecholamine-induced cAMP-PKA signaling in FOXC2 transgenic mice. Here we show that rolipram treatment of adipocytes from FOXC2 transgenic mice did not increase isoproterenol-induced cAMP accumulation to the same extent as in wild type cells. Accordingly, phosphodiesterase-4 (PDE4) activity was reduced by 75% and PDE4A5 protein expression reduced by 30-50% in FOXC2 transgenic WAT compared to wild type. Thus, reduced PDE4 activity in adipocytes from FOXC2 transgenic mice contributes to amplified beta-AR induced cAMP responses observed in these cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Constitutive activation of Galphas within forebrain neurons causes deficits in sensorimotor gating because of PKA-dependent decreases in cAMP.

Sensorimotor gating, the ability to automatically filter sensory information, is deficient in a number of psychiatric disorders, yet little is known of the biochemical mechanisms underlying this critical neural process. Previously, we reported that mice expressing a constitutively active isoform of the G-protein subunit Galphas (Galphas(*)) within forebrain neurons exhibit decreased gating, as measured by prepulse inhibition of acoustic startle (PPI). Here, to elucidate the biochemistry regulating sensorimotor gating and to identify novel therapeutic targets, we test the hypothesis that Galphas(*) causes PPI deficits via brain region-specific changes in cyclic AMP (cAMP) signaling. As predicted from its ability to stimulate adenylyl cyclase, we find here that Galphas(*) increases cAMP levels in the striatum. Suprisingly, however, Galphas(*) mice exhibit reduced cAMP levels in the cortex and hippocampus because of increased cAMP phosphodiesterase (cPDE) activity. It is this decrease in cAMP that appears to mediate the effect of Galphas(*) on PPI because Rp-cAMPS decreases PPI in C57BL/6J mice. Furthermore, the antipsychotic haloperidol increases both PPI and cAMP levels specifically in Galphas(*) mice and the cPDE inhibitor rolipram also rescues PPI deficits of Galphas(*) mice. Finally, to block potentially the pathway that leads to cPDE upregulation in Galphas(*) mice, we coexpressed the R(AB) transgene (a dominant-negative regulatory subunit of protein kinase A (PKA)), which fully rescues the reductions in PPI and cAMP caused by Galphas(*). We conclude that expression of Galphas(*) within forebrain neurons causes PPI deficits because of a PKA-dependent decrease in cAMP and suggest that cAMP PDE inhibitors may exhibit antipsychotic-like therapeutic effects.

Acoustic Stimulation↗

PubMed2006

Phosphodiesterase-4 influences the PKA phosphorylation status and membrane translocation of G-protein receptor kinase 2 (GRK2) in HEK-293beta2 cells and cardiac myocytes.

Membrane-recruitment of GRK2 (G-protein receptor kinase 2) provides a fundamental step in the desensitization process controlling GPCRs (G-protein-coupled receptors), such as the beta2AR (beta2-adrenergic receptor). In the present paper, we show that challenge of HEK-293beta2 [human embryonic kidney cells stably overexpressing the FLAG-tagged beta2AR-GFP (green fluorescent protein)] cells with the beta-adrenoceptor agonist, isoprenaline, causes GRK2 to become phosphorylated by PKA (cAMP-dependent protein kinase). This action is facilitated when cAMP-specific PDE4 (phosphodiesterase-4) activity is selectively inactivated, either chemically with rolipram or by siRNA (small interfering RNA)-mediated knockdown of PDE4B and PDE4D. PDE4-selective inhibition by rolipram facilitates the isoprenaline-induced membrane translocation of GRK2, phosphorylation of the beta2AR by GRK2, membrane translocation of beta-arrestin and internalization of beta2ARs. PDE4-selective inhibition also enhances the ability of isoprenaline to trigger the PKA phosphorylation of GRK2 in cardiac myocytes. In the absence of isoprenaline, rolipram-induced inhibition of PDE4 activity in HEK-293beta2 cells acts to stimulate PKA phosphorylation of GRK2, with consequential effects on GRK2 membrane recruitment and GRK2-mediated phosphorylation of the beta2AR. We propose that a key role for PDE4 enzymes is: (i) to gate the action of PKA on GRK2, influencing the rate of GRK2 phosphorylation of the beta2AR and consequential recruitment of beta-arrestin subsequent to beta-adrenoceptor agonist challenge, and (ii) to protect GRK2 from inappropriate membrane recruitment in unstimulated cells through its phosphorylation by PKA in response to fluctuations in basal levels of cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Spatial organisation of AKAP18 and PDE4 isoforms in renal collecting duct principal cells.

A plethora of stimuli including hormones and neurotransmitters mediate a rise of the cellular level of cAMP and thereby activation of protein kinase A (PKA). PKA phosphorylates and thereby modulates the activity of a wide range of cellular targets. It is now appreciated that different stimuli induce the activation of PKA at specific sites where the kinase phosphorylates particular substrates in close proximity. The tethering of PKA to cellular compartments is facilitated by A kinase-anchoring proteins (AKAPs). The incorporation of phosphodiesterases (PDEs) into AKAP-based signalling complexes provides gradients of cAMP that regulate PKA activity locally. An example for a process depending on compartmentalised cAMP/PKA signalling is the arginine-vasopressin (AVP)-mediated water reabsorption in renal collecting duct principal cells. Upon activation through AVP, PKA phosphorylates the water channel aquaporin-2 (AQP-2) located on intracellular vesicles. The phosphorylation triggers the redistribution of AQP2 to the plasma membrane. AKAP-anchored PKA has been shown to be involved in AQP2 shuttling. Here, AKAP18 isoforms and members of the PDE4 family of PDEs are shown to be differentially localised in renal principal cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

Hypoxia-induced remodelling of PDE4 isoform expression and cAMP handling in human pulmonary artery smooth muscle cells.

Human pulmonary artery smooth muscle cells (hPASM cells) express PDE4A10, PDE4A11, PDE4B2, PDE4C and PDE4D5 isoforms. Hypoxia causes a transient up-regulation of PDE4B2 that reaches a maximum after 7 days and sustained up-regulation of PDE4A10/11 and PDE4D5 over 14 days in hypoxia. Seven days in hypoxia increases both intracellular cAMP levels, protein kinase A (PKA) activity and activated, phosphorylated extracellular signal regulated kinase (pERK) but does not alter either PKA isoform expression or total cAMP phosphodiesterase-4 (PDE4) activity or cAMP phosphodiesterase-3 (PDE3) activity. Both the cyclooxygenase inhibitor, indomethacin and the ERK inhibitors, UO126 and PD980589 reverse the hypoxia-induced increase in intracellular cAMP levels back to those seen in normoxic hPASM cells. Challenge of normoxic hPASM cells with prostaglandin E(2) (PGE(2)) elevates cAMP to levels comparable to those seen in hypoxic cells but fails to increase intracellular cAMP levels in hypoxic hPASM cells. The adenylyl cyclase activator, forskolin increases cAMP levels in both normoxic and hypoxic hPASM cells to comparable elevated levels. Challenge of hypoxic hPASM cells with indomethacin attenuates total PDE4 activity whilst challenge with UO126 increases total PDE4 activity. We propose that the hypoxia-induced activation of ERK initiates a phospholipase A(2)/COX-driven autocrine effect whereupon PGE(2) is generated, causing the activation of adenylyl cyclase and increase in intracellular cAMP. Despite the hypoxia-induced increases in the expression of PDE4A10/11, PDE4B2 and PDE4D5 and activation of certain of these long PDE4 isoforms through PKA phosphorylation, we suggest that the failure to see any overall increase in PDE4 activity is due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. Such hypoxia-induced increase in expression of PDE4 isoforms known to interact with certain signalling scaffold proteins may result in alterations in compartmentalised cAMP signalling. The hypoxia-induced increase in cAMP may represent a compensatory protective mechanism against hypoxia-induced mitogens such as endothelin-1 and serotonin.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2006

A novel role for a Drosophila homologue of cGMP-specific phosphodiesterase in the active transport of cGMP.

cGMP was first discovered in urine, demonstrating that kidney cells extrude this cyclic nucleotide. Drosophila Malpighian tubules provide a model renal system in which a homologue of mammalian PDE (phosphodiesterase) 6 is expressed. In humans, this cG-PDE (cGMP-specific PDE) is specifically expressed in the retinal system, where it controls visual signal transduction. In order to gain insight into the functional role of DmPDE6 (Drosophila PDE6-like enzyme) in epithelial function, we generated transgenic animals with targeted expression of DmPDE6 to tubule Type I (principal) cells. This revealed localization of DmPDE6 primarily at the apical membranes. As expected, overexpression of DmPDE6 resulted in elevated cG-PDE activity and decreased tubule cGMP content. However, such targeted overexpression of DmPDE6 creates a novel phenotype that manifests itself in inhibition of the active transport and efflux of cGMP by tubules. This effect is specific to DmPDE6 action, as no effect on cGMP transport is observed in tubules from a bovine PDE5 transgenic line which display reduced rates of fluid secretion, an effect not seen in DmPDE6 transgenic animals. Specific ablation of DmPDE6 in tubule principal cells, via expression of a targeted DmPDE6 RNAi (RNA interference) transgene, conferred increased active transport of cGMP, confirming a direct role for DmPDE6 in regulating cGMP transport in tubule principal cells. Pharmacological inhibition of DmPDE6 in wild-type tubules using the cG-PDE inhibitor, zaprinast, similarly results in stimulated cGMP transport. We provide the first demonstration of a novel role for a cG-PDE in modulating cGMP transport and efflux.

Animals↗

PubMed2006

Intracellular targeting of phosphodiesterase-4 underpins compartmentalized cAMP signaling.

The phosphodiesterase-4 (PDE4) enzyme belongs to a family of cAMP-dependent phosphodiesterases that provide the major means of hydrolyzing and, thereby, inactivating the key intracellular second messenger, cAMP. As such, PDE4s are central to the regulation of many diverse signaling processes that allow cells to respond to external stimuli. Four genes (4A, 4B, 4C, and 4D) encode around 20 distinct isoform members of the PDE4 family. Each isoform is characterized by a unique N-terminal region. PDE4s are multidomain metallohydrolases with each domain serving particular roles allowing them to be targeted to varying regions and organelles of intracellular space and regulated in distinct fashions by phosphorylation and protein-protein interaction. Although identical in catalytic function, each isoform locates to distinct regions within the cell so as to create and manage spatially distinct pools of cAMP. The multiplicity of partners associating with members of the four gene PDE4 family places these enzymes in key regulatory positions, permitting them to channel complex biological signals via fundamental signaling cohorts such as G-protein-coupled receptors (GPCRs), arrestins, A-kinase-anchoring proteins (AKAPs), and tyrosyl family kinases. The cAMP cascade has long been linked to cellular growth and embryogenesis and with this comes the implication that PDE4 may play considerable roles in the regulation of progeny development in maturing cells and tissues.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Compartmentalized phosphodiesterase-2 activity blunts beta-adrenergic cardiac inotropy via an NO/cGMP-dependent pathway.

beta-Adrenergic signaling via cAMP generation and PKA activation mediates the positive inotropic effect of catecholamines on heart cells. Given the large diversity of protein kinase A targets within cardiac cells, a precisely regulated and confined activity of such signaling pathway is essential for specificity of response. Phosphodiesterases (PDEs) are the only route for degrading cAMP and are thus poised to regulate intracellular cAMP gradients. Their spatial confinement to discrete compartments and functional coupling to individual receptors provides an efficient way to control local [cAMP]i in a stimulus-specific manner. By performing real-time imaging of cyclic nucleotides in living ventriculocytes we identify a prominent role of PDE2 in selectively shaping the cAMP response to catecholamines via a pathway involving beta3-adrenergic receptors, NO generation and cGMP production. In cardiac myocytes, PDE2, being tightly coupled to the pool of adenylyl cyclases activated by beta-adrenergic receptor stimulation, coordinates cGMP and cAMP signaling in a novel feedback control loop of the beta-adrenergic pathway. In this, activation of beta3-adrenergic receptors counteracts cAMP generation obtained via stimulation of beta1/beta2-adrenoceptors. Our study illustrates the key role of compartmentalized PDE2 in the control of catecholamine-generated cAMP and furthers our understanding of localized cAMP signaling.

Adenine↗

PubMed2005

DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling.

The disrupted in schizophrenia 1 (DISC1) gene is a candidate susceptibility factor for schizophrenia, but its mechanistic role in the disorder is unknown. Here we report that the gene encoding phosphodiesterase 4B (PDE4B) is disrupted by a balanced translocation in a subject diagnosed with schizophrenia and a relative with chronic psychiatric illness. The PDEs inactivate adenosine 3',5'-monophosphate (cAMP), a second messenger implicated in learning, memory, and mood. We show that DISC1 interacts with the UCR2 domain of PDE4B and that elevation of cellular cAMP leads to dissociation of PDE4B from DISC1 and an increase in PDE4B activity. We propose a mechanistic model whereby DISC1 sequesters PDE4B in resting cells and releases it in an activated state in response to elevated cAMP.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

Keynote review: phosphodiesterase-4 as a therapeutic target.

Cyclic AMP (cAMP) is a key second messenger in all cells. It is compartmentalized within cells and its levels are controlled, as a result of spatially discrete signaling cassettes controlling its generation, detection and degradation. Underpinning compartmentalized cAMP signaling are approximately 20 members of the phosphodiesterase-4 (PDE4) family. The selective inhibition of this family generates profound, functional effects and PDE4 inhibitors are currently under development to provide potential, novel therapeutics for the treatment of inflammatory diseases, such as asthma, chronic obstructive pulmonary disease and psoriasis, as well as treating depression and serving as cognitive enhancers. Here, we delineate the range of PDE4 isoforms, their role in signaling, their structural biology and related preclinical and clinical pharmacology.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

RNA silencing identifies PDE4D5 as the functionally relevant cAMP phosphodiesterase interacting with beta arrestin to control the protein kinase A/AKAP79-mediated switching of the beta2-adrenergic receptor to activation of ERK in HEK293B2 cells.

PDE4B and PDE4D provide >90% of PDE4 cAMP phosphodiesterase activity in human embryonic kidney (HEK293B2) cells. Their selective small interference RNA (siRNA)-mediated knockdown potentiates isoprenaline-stimulated protein kinase A (PKA) activation. Whereas endogenous PDE4D co-immunoprecipitates with beta arrestin, endogenous PDE4B does not, even upon PDE4D knockdown. Ectopic overexpression of PDE4B2 confers co-immunoprecipitation with beta arrestin. Knockdown of PDE4D, but not PDE4B, amplifies isoprenaline-stimulated phosphorylation of the beta2-adrenergic receptor (beta2-AR) by PKA and activation of extracellular signal-regulated kinase (ERK) through G(i). Isoform-selective knockdown identifies PDE4D5 as the functionally important species regulating isoprenaline stimulation of both these processes. Ht31-mediated disruption of the tethering of PKA to AKAP scaffold proteins attenuates isoprenaline activation of ERK, even upon PDE4D knockdown. Selective siRNA-mediated knockdown identifies AKAP79, which is constitutively associated with the beta2-AR, rather than isoprenaline-recruited gravin, as being the functionally relevant AKAP in this process. Isoprenaline-stimulated membrane recruitment of PDE4D is ablated upon beta arrestin knockdown. A mutation that compromises interactions with beta arrestin prevents catalytically inactive PDE4D5 from performing a dominant negative role in potentiating isoprenaline-stimulated ERK activation. Beta arrestin-recruited PDE4D5 desensitizes isoprenaline-stimulated PKA phosphorylation of the beta2-AR and the consequential switching of its signaling to ERK. The ability to observe a cellular phenotype upon PDE4D5 knockdown demonstrates that other PDE4 isoforms, expressed at endogenous levels, are unable to afford rescue in HEK293B2 cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2005

The long and short of vascular smooth muscle phosphodiesterase-4 as a putative therapeutic target.

In this issue, Tilley and Maurice (p. 596) show that differentiation of vascular smooth muscle cells to a proliferative phenotype is associated with a profound up-regulation of specific phosphodiesterase-4 (PDE4) isoforms because of increased histone acetylation. The increased PDE4 activity is seen as preventing cAMP from inhibiting the enhanced proliferation, migration, and production of extracellular matrix seen in activated VSMC. This Perspective examines the proposal that selective inhibition of PDE4D1/2 could find use in adjunctive pharmacotherapy after percutaneous coronary interventions and, in addition, discusses the recent genetic evidence that PDE4D7 may provide a therapeutic target in stroke.

3',5'-Cyclic-AMP Phosphodiesterases↗