Long-term regulation of cyclic nucleotide phosphodiesterase type 3B and 4 in 3T3-L1 adipocytes

Phosphodiesterase 3B (PDE3B), is known to play an important role in acute insulin and cAMP-mediated regulation of lipid metabolism, and PDE4 are the main PDE types expressed in adipocytes. Here, we show that members of all PDE4 isoforms are expressed in 3T3-L1 and primary mouse adipocytes. Long-term treatment of 3T3-L1 adipocytes with insulin induced up-regulation of PDE3B and PDE4D in a phosphatidylinositol 3-kinase-dependent manner whereas long-term treatment with beta-adrenergic agonists induced down-regulation of PDE3B and up-regulation of PDE4D. Thus, PDE3B and PDE4D can be added to the list of genes regulated by insulin and cAMP-increasing hormones. Altered expression of PDE3B and PDE4D in response to long-term treatment with insulin and catecholamines may contribute to altered regulation of metabolism in diabetes.     

Structure-based design of selective phosphodiesterase 4B inhibitors based on ginger phenolic compounds

Phosphodiesterase 4 (PDE4) has been established as a drug target for inflammatory diseases of respiratory tract like asthma and chronic obstructive pulmonary disease. The selective inhibitors of PDE4B, a subtype of PDE4, are devoid of adverse effects like nausea and vomiting commonly associated with non-selective PDE4B inhibitors. This makes the development of PDE4B subtype selective inhibitors a desirable research goal. Thus, in the present study, molecular docking, molecular dynamic simulations and binding free energy were performed to explore potential selective PDE4B inhibitors based on ginger phenolic compounds. The results of docking studies indicate that some of the ginger phenolic compounds demonstrate higher selective PDE4B inhibition than existing selective PDE4B inhibitors. Additionally, 6-gingerol showed the highest PDE4B inhibitory activity as well as selectivity. The comparison of binding mode of PDE4B/6-gingerol and PDE4D/6-gingerol complexes revealed that 6-gingerol formed additional hydrogen bond and hydrophobic interactions with active site and control region 3 (CR3) residues in PDE4B, which were primarily responsible for its PDE4B selectivity. The results of binding free energy demonstrated that electrostatic energy is the primary factor in elucidating the mechanism of PDE4B inhibition by 6-gingerol. Dynamic cross-correlation studies also supported the results of docking and molecular dynamics simulation. Finally, a small library of molecules were designed based on the identified structural features, majority of designed molecules showed higher PDE4B selectivity than 6-gingerol. These results provide important structural features for designing new selective PDE4B inhibitors as anti-inflammatory drugs and promising candidates for synthesis and pre-clinical pharmacological investigations.     

Ndel1 alters its conformation by sequestering cAMP-specific phosphodiesterase-4D3 (PDE4D3) in a manner that is dynamically regulated through Protein Kinase A (PKA)

The involvement of the Nuclear distribution element-like (Ndel1; Nudel) protein in the recruitment of the dynein complex is critical for neurodevelopment and potentially important for neuronal disease states. The PDE4 family of phosphodiesterases specifically degrades cAMP, an important second messenger implicated in learning and memory functions. Here we show for the first time that Ndel1 can interact directly with PDE4 family members and that the interaction of Ndel1 with the PDE4D3 isoform is uniquely disrupted by elevation of intracellular cAMP levels. While all long PDE4 isoforms are subject to stimulatory PKA phosphorylation within their conserved regulatory UCR1 domain, specificity for release of PDE4D3 is conferred due to the PKA-dependent phosphorylation of Ser13 within the isoform-specific, unique amino-terminal domain of PDE4D3. Scanning peptide array analyses identify a common region on Ndel1 for PDE4 binding and an additional region that is unique to PDE4D3. The common site lies within the stutter region that links the second coiled-coil region to the unstable third coiled-coil regions of Ndel1. The additional binding region unique to PDE4D3 penetrates into the start of the third coiled-coil region that can undergo tail-to-tail interactions between Ndel1 dimers to form a 4 helix bundle. We demonstrate Ndel1 self-interaction in living cells using a BRET approach with luciferase- and GFP-tagged forms of Ndel1. BRET assessed Ndel1–Ndel1 self-interaction is amplified through the binding of PDE4 isoforms. For PDE4D3 this effect is ablated upon elevation of intracellular cAMP due to PKA-mediated phosphorylation at Ser13, while the potentiating effects of PDE4B1 and PDE4D5 are resistant to cAMP elevation. PDE4D long isoforms and Ndel1 show a similar sub-cellular distribution in hippocampus and cortex and locate to post-synaptic densities. We show that Ndel1 sequesters EPAC, but not PKA, in order to form a cAMP signalling complex. We propose that a key function of the Ndel1 signalling scaffold is to signal through cAMP by sequestering EPAC, whose activity may thus be specifically regulated by sequestered PDE4 that also stabilizes Ndel1–Ndel1 self-interaction. In the case of PDE4D3, its association with Ndel1 is dynamically regulated by PKA input through its ability to phosphorylate Ser13 in the unique N-terminal region of this isoform, triggering the specific release of PDE4D3 from Ndel1 when cAMP levels are elevated. We propose that Ser13 may act as a redistribution trigger in PDE4D3, allowing it to dynamically re-shape cAMP gradients in distinct intracellular locales upon its phosphorylation by PKA.     

Discovery of triazines as potent,selective and orally active PDE4 inhibitors

Expanding on HTS hit 4 afforded a series of [1,3,5]triazine derivatives as novel PDE4 inhibitors. The SAR development and optimization process with the emphasis on ligand efficiency and physicochemical properties led to the discovery of compound 44 as a potent, selective and orally active PDE4 inhibitor.     

Serpin B3/B4, activated by STAT3, promote survival of squamous carcinoma cells

Persistently activated STAT3 contributes to cell survival in many different human cancers. Cancer cell secretion of IL-6 is a frequent basis for persistent STAT3 activation; we show that antibodies against IL-6 or gp-130, the signaling unit of the IL-6 receptor, can abruptly remove persistently activated STAT3 causing prompt disappearance of cysteine proteases of serpin B3/B4 mRNAs, known as squamous cell carcinoma antigens 1 and 2. STAT3 occupies the promoter of serpin B3/B4 before removal and siRNA removal of B3/B4 mRNA caused cell death in HN13 head and neck cancer cells. Thus persistently activated STAT3 is a required part of the continuous activation of B3/B4 genes, which protects tumor cells from dying.     

Inhibition of phosphodiesterase activity, airway inflammation and hyperresponsiveness by PDE4 inhibitor and glucocorticoid in a murine model of allergic asthma

Phosphodiesterase 4 (PDE4) isozyme plays important roles in inflammatory and immunomodulatory cells. In this study, piclamilast, a selective PDE4 inhibitor, was used to investigate the role of PDE4 in respiratory function and inflammation in a murine asthma model. Sensitized mice were challenged with aerosolized ovalbumin for 7 days, piclamilast (1, 3 and 10 mg/kg) and dexamethasone (2 mg/kg) were orally administered once daily during the period of challenge. Twenty-four hours after the last challenge, airway hyperresponsiveness to methacholine was determined by whole-body plethysmography, airway inflammation and mucus secretion by histomorphometry, pulmonary cAMP-PDE activity by HPLC, cytokine levels in bronchoalveolar lavage fluid and their mRNA expression in lung by ELISA and RT-PCR, respectively. In control mice, significant induction of cAMP-PDE activity was parallel to the increases of hyperresponsiveness, inflammatory cells, cytokine levels, mRNA expression as well as goblet cell hyperplasia. However, piclamilast dose-dependently and significantly improved airway resistance and dynamic compliance, and the maximal effect was similar to that of dexamethasone. Piclamilast treatment dose-dependently and significantly prevented the increase in inflammatory cell number and goblet cell hyperplasia, as well as production of cytokines, including eotaxin, TNFalpha and IL-4. Piclamilast exerted a weaker inhibitory effect than dexamethasone on eosinophils and neutrophils, had no effect on lymphocyte accumulation. Moreover, piclamilast inhibited up-regulation of cAMP-PDE activity and cytokine mRNA expression; the maximal inhibition of cAMP-PDE was greater than that exerted by dexamethasone, and was similar to dexamethasone on cytokine mRNA expression. This study suggests that inhibition of PDE4 by piclamilast robustly improves the pulmonary function, airway inflammation and goblet cell hyperplasia in murine allergenic asthma.     

The mAKAP signaling complex: integration of cAMP, calcium, and MAP kinase signaling pathways

Following its production by adenylyl cyclases, the second messenger cAMP is in involved in pleiotrophic signal transduction. The effectors of cAMP include the cAMP-dependent protein kinase (PKA), the guanine nucleotide exchange factor Epac (exchange protein activated by cAMP), and cAMP-dependent ion channels. In turn, cAMP signaling is attenuated by phosphodiesterase-catalyzed degradation. The association of cAMP effectors and the enzymes that regulate cAMP concentration into signaling complexes helps to explain the differential signaling initiated by members of the G(s)-protein coupled receptor family. The signal transduction complex formed by the scaffold protein mAKAP (muscle A kinase-anchoring protein) at the nuclear envelope of both striated myocytes and neurons contains three cAMP-binding proteins, PKA, Epac1, and the phosphodiesterase PDE4D3. In addition, the mAKAP complex also contains components of the ERK5 MAP kinase signaling pathway, the calcium release channel ryanodine receptor and the phosphatases PP2A as well as calcineurin. Analysis of the mAKAP complex illustrates how a macromolecular complex can serve as a node in the intracellular signaling network of cardiac myocytes to integrate multiple cAMP signals with those of calcium and MAP kinases to regulate the hypertrophic actions of several hormones.     

Glycosynaptic microdomains controlling tumor cell phenotype through alteration of cell growth, adhesion, and motility

Glycosphingolipids (GSLs) GM3 (NeuAcα3Galβ4Glcβ1Cer) and GM2 (GalNAcβ4[NeuAcα3]Galβ4Glcβ1Cer) inhibit (i) cell growth through inhibition of tyrosine kinase associated with growth factor receptor (GFR), (ii) cell adhesion/motility through inhibition of integrin-dependent signaling via Src kinases, or (iii) both cell growth and motility by blocking “cross-talk” between integrins and GFRs. These inhibitory effects are enhanced when GM3 or GM2 are in complex with specific tetraspanins (TSPs) (CD9, CD81, CD82). Processes (i)-(iii) occur through specific organization of GSLs with key molecules (TSPs, caveolins, GFRs, integrins) in the glycosynaptic microdomain. Some of these processes are shared with epithelial-mesenchymal transition induced by TGFβ or under hypoxia, particularly that associated with cancer progression.     

Spatial restriction of alpha4 integrin phosphorylation regulates lamellipodial stability and alpha4beta1-dependent cell migration

Integrins coordinate spatial signaling events essential for cell polarity and directed migration. Such signals from alpha4 integrins regulate cell migration in development and in leukocyte trafficking. Here, we report that efficient alpha4-mediated migration requires spatial control of alpha4 phosphorylation by protein kinase A, and hence localized inhibition of binding of the signaling adaptor, paxillin, to the integrin. In migrating cells, phosphorylated alpha4 accumulated along the leading edge. Blocking alpha4 phosphorylation by mutagenesis or by inhibition of protein kinase A drastically reduced alpha4-dependent migration and lamellipodial stability. alpha4 phosphorylation blocks paxillin binding in vitro; we now find that paxillin and phospho-alpha4 were in distinct clusters at the leading edge of migrating cells, whereas unphosphorylated alpha4 and paxillin colocalized along the lateral edges of those cells. Furthermore, enforced paxillin association with alpha4 inhibits migration and reduced lamellipodial stability. These results show that topographically specific integrin phosphorylation can control cell migration and polarization by spatial segregation of adaptor protein binding.     

Essential role of phosphatidylinositol 3-kinase in insulin-induced activation and phosphorylation of the cGMP-inhibited cAMP phosphodiesterase in rat adipocytes studies using the selective inhibitor wortmannin

Incubation of rat adipocytes with wortmannin, a potent and selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, completely blocked the antilipolytic action of insulin (IC₅₀≈ 100 nM), the insulin-induced activation and phosphorylation of cGMP-inhibited cAMP phosphodiesterase (cGI-PDE) as well as the activation of the insulin-stimulated cGI-PDE kinase (IC₅₀≈ 10–30 nM). No direct effects of the inhibitor on the insulin-stimulated cGI-PDE kinase, the cGI-PDE and the hormone-sensitive lipase were observed. These data suggest that activation of PI 3-kinase upstream of the insulin-stimulated cGI-PDE kinase in the antilipolytic insulin signalchain has an essential role for insulin-induced cGI-PDE activation/ phosphorylation and anti-lipolysis.     

Overexpression of exchange protein directly activated by cAMP-1 (EPAC1) attenuates bladder cancer cell migration

Exchange protein directly activated by cAMP (EPAC) is a mediator of a cAMP signaling pathway that is independent of protein kinase A. EPAC has two isoforms (EPAC1 and EPAC2) and is a cAMP-dependent guanine nucleotide exchange factor for the small GTPases, Rap1 and Rap2. Recent studies suggest that EPAC1 has both positive and negative influences on cancer and is involved in cell proliferation, apoptosis, migration and metastasis. We report that EPAC1 and EPAC2 expression levels were significantly lower in bladder cancer tissue than in normal bladder tissue. In addition, bladder cancer cell lines showed reduced EPAC1 mRNA expression. Furthermore, EPAC1 overexpression in bladder cancer cell lines induced morphologic changes and markedly suppressed cell migration without affecting cell viability. The overexpressed EPAC1 preferentially localized at cell-cell interfaces. In conclusion, reduced EPAC1 expression in bladder tumors and poor migration of EPAC1-overexpressing cells implicate EPAC1 as an inhibitor of bladder cancer cell migration.     

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low k(cat) despite a tight K(m). Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X-ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site-directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had k(cat)/K(m) less than 10% of the wild-type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3- to 12-fold increase of K(m) for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9- to 200-fold increase of K(i) for cGMP in comparison to the wild-type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.     

Inhibitory effects of dynorphin 3-14 on the lipopolysaccharide-induced toll-like receptor 4 signalling pathway

Dynorphin 1-17 (DYN 1-17) is biotransformed rapidly to a range of fragments in rodent inflamed tissue with dynorphin 3-14 (DYN 3-14) being the most stable and prevalent. DYN 1-17 has been shown previously to be involved in the regulation of inflammatory response following tissue injury, in which the biotransformation fragments of DYN 1-17 may possess similar features. This study investigated the effects of DYN 3-14 on lipopolysaccharide (LPS)-induced nuclear factor-kappaB/p65 (NF-κB/p65) nuclear translocation and the release of pro-inflammatory cytokines interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in differentiated THP-1 cells. Treatment with DYN 3-14 (10 nM) resulted in 35% inhibition of the LPS-induced nuclear translocation of NF-κB/p65. Furthermore, DYN 3-14 modulated both IL-1β and TNF-α release; inhibiting IL-1β and paradoxically augmenting TNF-α release in a concentration-independent manner. A number of opioids have been implicated in the modulation of the toll-like receptor 4 (TLR4), highlighting the complexity of their immunomodulatory effects. To determine whether DYN 3-14 modulates TLR4, HEK-Blue™⁻hTLR4 cells were stimulated with LPS in the presence of DYN 3-14. DYN 3-14 (10 μM) inhibited TLR4 activation in a concentration-dependent fashion by suppressing the LPS signals around 300-fold lower than LPS-RS, a potent TLR4 antagonist. These findings indicate that DYN 3-14 is a potential TLR4 antagonist that alters cellular signaling in response to LPS and cytokine release, implicating a role for biotransformed endogenous opioid peptides in immunomodulation.     

Isolation,characterization and molecular 3D model of human GDE4, a novel membrane protein containing glycerophosphodiester phosphodiesterase domain

As a transmembrane protein family, glycerophosphodiester phosphodiesterase (GDPD/GDE) catalyzes the hydrolysis of deacylated glycerophospholipids to glycerol phosphate and alcohol. To date, seven mammalian GDEs have been virtually cloned or predicted by bioinformatics analysis, however, GDE4 has not been molecular isolated and characterized in mammal. Here we report molecular cloning of human GDE4 encoding cDNA sequence, which is 945 base pairs long encoding a 314-amino acid protein with 2 transmembrane regions and a GDE motif. The human GDE1 gene is located on chromosome 19q22 and contains ten exons and nine introns. A molecular 3-D model provides the first structural information of human GDE4 and suggests a triose-phosphate-isomerase barrel core as typically found in bacterial GDPDs. Furthermore, a model of the putative catalytic residues highlights that the individual core residues Glu72, Asp74, and His87 are crucial to maintaining GDE4 catalytic activity. Western blotting shows that human GDE4 is a 36 kDa protein. Subcellular localization of GDE4 tagged with enhanced green fluorescence protein is in the cytoplasm, especially accumulated in the perinuclear region and the cell periphery. Moreover, over-expression of GDE4 did not induce neurite formation or change cell morphology. These results indicate GDE4 protein is a member of the GDE family and suggest it may play different roles from other members of GDE family.