Vincent C. Manganiello

Foxp3-dependent programme of regulatory T-cell differentiation

Regulatory CD4+ T cells (Tr cells), the development of which is critically dependent on X-linked transcription factor Foxp3 (forkhead box P3), prevent self-destructive immune responses. Despite its important role, molecular and functional features conferred by Foxp3 to Tr precursor cells remain unknown. It has been suggested that Foxp3 expression is required for both survival of Tr precursors as well as their inability to produce interleukin (IL)-2 and independently proliferate after T-cell-receptor engagement, raising the possibility that such ‘anergy’ and Tr suppressive capacity are intimately linked. Here we show, by dissociating Foxp3-dependent features from those induced by the signals preceding and promoting its expression in mice, that the latter signals include several functional and transcriptional hallmarks of Tr cells. Although its function is required for Tr cell suppressor activity, Foxp3 to a large extent amplifies and fixes pre-established molecular features of Tr cells, including anergy and dependence on paracrine IL-2. Furthermore, Foxp3 solidifies Tr cell lineage stability through modification of cell surface and signalling molecules, resulting in adaptation to the signals required to induce and maintain Tr cells. This adaptation includes Foxp3-dependent repression of cyclic nucleotide phosphodiesterase 3B, affecting genes responsible for Tr cell homeostasis.

Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKβ-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.

Structure, localization, and regulation of cGMP-inhibited phosphodiesterase (PDE3)

cAMP and cGMP mediate biological responses initiated by diverse extracellular signals. By catalyzing hydrolysis of the 39–59phosphodiester bond of cyclic nucleotides, cyclic nucleotide phosphodiesterases (PDEs) regulate intracellular concentrations and effects of these second messengers. PDEs include a large group of structurally related enzymes (reviewed in Refs. 1–3). These enzymes belong to at least seven related gene families (PDEs 1–7) (Fig. 1), which differ in their primary structures, affinities for cAMP and cGMP, responses to specific effectors, sensitivities to specific inhibitors, and mechanisms of regulation (1–3). Most families are comprised of more than one gene; 14 different PDE genes have been identified. Within different families, tissue-specific mRNAs are generated from the same gene by the use of different transcription initiation sites or by alternative mRNA splicing. Although some aspects of different PDE families will be discussed, this review emphasizes the PDE3 family, including structure-function information and regulation of the adipocyte PDE3, which plays a key role in the antilipolytic action of insulin. Mammalian PDEs share a common structural organization, with a conserved catalytic core (;270 amino acids) usually located in the C-terminal half (Fig. 1) (4). This region is much more similar within an individual PDE family (.80% amino acid identity) than between different PDE families (;25–40% identity) (1–4). The catalytic core is thought to contain common structural elements important for hydrolysis of the cyclic nucleotide phosphodiester bond, as well as family-specific determinants responsible for differences in substrate affinities and inhibitor sensitivities among the different gene families. It contains a PDE-specific sequence motif, HD(X)2H(X4)N, and two consensus Zn -binding domains, the second of which overlaps the PDE motif (3, 5). PDE5 contains tightly bound Zn, which supports catalytic activity (5). The precise role of Zn or other divalent cations in catalytic function of other PDEs has not been defined. Mutagenesis of the first histidine of the PDE sequence motif abolished activity of a recombinant PDE4 expressed in Escherichia coli (6). Histidineand sulfhydryl-modifying reagents inhibited PDE3 activity (7). The widely divergent N-terminal portions of PDEs (Fig. 1) contain determinants that confer regulatory properties specific to the different gene families, e.g. calmodulin-binding domains (PDE1); two non-catalytic cyclic nucleotide-binding domains (PDEs 2, 5, and 6); N-terminal membrane-targeting (PDE4) or hydrophobic membrane-association (PDE3) domains; and calmodulin (PDE1)-, cyclic AMP (PDEs 1, 3, and 4)-, and cGMP (PDE5)-dependent protein kinase phosphorylation sites, etc. (Fig. 1) (1–3). Most cells contain representatives of several PDE families in different amounts, proportions, and subcellular locations (1–3). In some instances a specific PDE regulates a unique cellular function, e.g. photoreceptor PDE6 in cGMP-dependent initiation of visual transduction. In individual cells, different PDEs, with their different responses to regulatory signals, participate in integrating multiple inputs in the complex modulation and termination of cyclic nucleotide signals and responses, e.g. their magnitude and duration, their functional and spatial compartmentation, and their attenuation by short-term feedback or long-term desensitization.

Advances in targeting cyclic nucleotide phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) catalyse the hydrolysis of cyclic AMP and cyclic GMP, thereby regulating the intracellular concentrations of these cyclic nucleotides, their signalling pathways and, consequently, myriad biological responses in health and disease. Currently, a small number of PDE inhibitors are used clinically for treating the pathophysiological dysregulation of cyclic nucleotide signalling in several disorders, including erectile dysfunction, pulmonary hypertension, acute refractory cardiac failure, intermittent claudication and chronic obstructive pulmonary disease. However, pharmaceutical interest in PDEs has been reignited by the increasing understanding of the roles of individual PDEs in regulating the subcellular compartmentalization of specific cyclic nucleotide signalling pathways, by the structure-based design of novel specific inhibitors and by the development of more sophisticated strategies to target individual PDE variants.

Regulation and function of the cyclic nucleotide phosphodiesterase (PDE3) gene family

Publisher Summary This chapter discusses some general information about cyclic nucleotide phosphodiesterases (PDEs). It also discusses the PDE3 gene family, emphasizing the molecular biology, structure/function relationships, and cellular regulation and functional roles of PDE3s, as well as physiological/pharmacological actions, therapeutic applications, and potential benefits of PDE3 inhibitors. The major cause of concern in the use of PDE3 inhibitors as therapeutic agents is the potential for increased mortality in patients with known heart disease. Although caution is certainly warranted in this context, conclusions should not be indiscriminately applied to all PDE3 inhibitors. The pharmacological profiles of newer PDE3 inhibitors differ from those of the PDE3 inhibitors used in earlier heart failure clinical trials. Although milrinone and cilostazol are similar in potency as inhibitors of PDE3, milrinone had greater effects than cilostazol on increasing both cyclic adenosine monophosphate (cAMP) and contractility in isolated rabbit cardiomyocytes. The ability to target PDE3 inhibitors to specific isoforms in specific intracellular compartments and/or specific cells may be critical for improvement in efficacy and safety. The acute benefits and chronic adverse actions of PDE3 inhibitors in patients, with heart failure, may result from the phosphorylation of different substrates of Protein kinase A (PKA) in different intracellular compartments. Newer PDE3 inhibitors that target a specific isoform in the appropriate compartment could potentially confer beneficial hemodynamic effects without adverse effects on mortality.

Distinctive anatomical patterns of gene expression for cGMP-inhibited cyclic nucleotide phosphodiesterases.

Type III cGMP-inhibited phosphodiesterases (PDE3s) play important roles in hormonal regulation of lipolysis, platelet aggregation, myocardial contractility, and smooth muscle relaxation. We have recently characterized two PDE3 subtypes (PDE3A and PDE3B) as products of distinct but related genes. To elucidate their biological roles, in this study we compare cellular patterns of gene expression for these two enzymes during rat embryonic and postnatal development using in situ hybridization. PDE3B [corrected] mRNA is abundant in adipose tissue and is also expressed in hepatocytes throughout development. This mRNA is also highly abundant in embryonic neuroepithelium including the neural retina, but expression is greatly reduced in the mature nervous system. Finally, PDE3B [corrected] mRNA is localized in spermatocytes and renal collecting duct epithelium in adult rats. PDE3B mRNA is highly expressed in the cardiovascular system, including myocardium and arterial and venous smooth muscle, throughout development. It is also abundant in bronchial, genitourinary and gastrointestinal smooth muscle and epithelium, megakaryocytes, and oocytes. PDE3A [corrected] mRNA demonstrates a complex, developmentally regulated pattern of gene expression in the central nervous system. In summary, the two different PDE3s show distinctive tissue-specific patterns of gene expression suggesting that PDE3B [corrected] is involved in hormonal regulation of lipolysis and glycogenolysis, while regulation of myocardial and smooth muscle contractility appears to be a function of PDE3A [corrected]. In addition, the present findings suggest previously unsuspected roles for these enzymes in gametogenesis and neural development.

Protein phosphatase 2A is the main phosphatase involved in the regulation of protein kinase B in rat adipocytes.

In adipocytes, protein kinase B (PKB) has been suggested to be the enzyme that phosphorylates phosphodiesterase 3B (PDE3B), a key enzyme in insulins antilipolytic signalling pathway. In order to screen for PKB phosphatases, adipocyte homogenates were fractionated using ion-exchange chromatography and analysed for PKB phosphatase activities. PKB phosphatase activity eluted as one main peak, which coeluted with serine/threonine phosphatases (PP)2A. In addition, adipocytes were incubated with inhibitors of PP. Incubation of adipocytes with 1 microM okadaic acid inhibited PP2A by 75% and PP1 activity by only 17%, while 1 microM tautomycin inhibited PP1 activity by 54% and PP2A by only 7%. Okadaic acid, but not tautomycin, induced the activation of both PKBalpha and PKBbeta. Finally, PP2A subunits were found in several subcellular compartments, including plasma membranes (PM) where the phosphorylation of PKB is thought to occur. In summary, our results suggest that PP2A is the principal phosphatase that dephosphorylates PKB in adipocytes.

Alterations in regulation of energy homeostasis in cyclic nucleotide phosphodiesterase 3B–null mice

Cyclic nucleotide phosphodiesterase 3B (PDE3B) has been suggested to be critical for mediating insulin/IGF-1 inhibition of cAMP signaling in adipocytes, liver, and pancreatic beta cells. In Pde3b-KO adipocytes we found decreased adipocyte size, unchanged insulin-stimulated phosphorylation of protein kinase B and activation of glucose uptake, enhanced catecholamine-stimulated lipolysis and insulin-stimulated lipogenesis, and blocked insulin inhibition of catecholamine-stimulated lipolysis. Glucose, alone or in combination with glucagon-like peptide-1, increased insulin secretion more in isolated pancreatic KO islets, although islet size and morphology and immunoreactive insulin and glucagon levels were unchanged. The beta(3)-adrenergic agonist CL 316,243 (CL) increased lipolysis and serum insulin more in KO mice, but blood glucose reduction was less in CL-treated KO mice. Insulin resistance was observed in KO mice, with liver an important site of alterations in insulin-sensitive glucose production. In KO mice, liver triglyceride and cAMP contents were increased, and the liver content and phosphorylation states of several insulin signaling, gluconeogenic, and inflammation- and stress-related components were altered. Thus, PDE3B may be important in regulating certain cAMP signaling pathways, including lipolysis, insulin-induced antilipolysis, and cAMP-mediated insulin secretion. Altered expression and/or regulation of PDE3B may contribute to metabolic dysregulation, including systemic insulin resistance.

Insulin-induced phosphorylation and activation of phosphodiesterase 3B in rat adipocytes: possible role for protein kinase B but not mitogen-activated protein kinase or p70 S6 kinase.

Insulin stimulation of adipocytes results in serine phosphorylation/activation of phosphodiesterase 3B (PDE 3B) and activation of a kinase that phosphorylates PDE 3B in vitro, key events in the antilipolytic action of this hormone. We have investigated the role for p70 S6 kinase, mitogen-activated protein kinases (MAP kinases), and protein kinase B (PKB) in the insulin signaling pathway leading to phosphorylation/activation of PDE 3B in adipocytes. Insulin stimulation of adipocytes resulted in increased activity of p70 S6 kinase, which was completely blocked by pretreatment with rapamycin. However, rapamycin had no effect on the insulin-induced phosphorylation/activation of PDE 3B or the activation of the kinase that phosphorylates PDE 3B. Stimulation of adipocytes with insulin or phorbol myristate acetate induced activation of MAP kinases. Pretreatment of adipocytes with the MAP kinase kinase inhibitor PD 98059 was without effect on the insulin-induced activation of PDE 3B. Furthermore, phorbol myristate acetate stimulation did not result in phosphorylation/activation of PDE 3B or activation of the kinase that phosphorylates PDE 3B. Using Mono Q and Superdex chromatography, the kinase that phosphorylates PDE 3B was found to co-elute with PKB, but not with p70 S6 kinase or MAP kinases. Furthermore, both PKB and the kinase that phosphorylates PDE 3B were found to translocate to membranes in response to peroxovanadate stimulation of adipocytes in a wortmannin-sensitive way. Whereas these results suggest that p70 S6 kinase and MAP kinases are not involved in the insulin-induced phosphorylation/activation of PDE 3B in rat adipocytes, they are consistent with PKB being the kinase that phosphorylates PDE 3B.

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.