In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca²⁺. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the “off signal” afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5′NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca²⁺ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.     

Development of Phosphodiesterase–Protein-Kinase Complexes as Novel Targets for Discovery of Inhibitors with Enhanced Specificity

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE–PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE–PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems—cAMP-specific PDE8–PKAR, cGMP-specific PDE5–PKG, and dual-specificity RegA–R_D complexes—and ranked inhibitors according to their inhibition potency. Targeting PDE–PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.     

Phosphodiesterases Expression during Murine Cardiac Development

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.     

Cellular Redox Metabolism Is Modulated by the Distinct Localization of Cyclic Nucleotide Phosphodiesterase 5A Isoforms

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a family of evolutionarily conserved cAMP and/or cGMP hydrolyzing enzymes, components of transduction pathways regulating crucial aspects of cell life. Among them, cGMP-specific PDE5—being a regulator of vascular smooth muscle contraction—is the molecular target of several drugs used to treat erectile dysfunction and pulmonary hypertension. Production of full-length murine PDE5A isoforms in the milk-yeast Kluyveromyces lactis showed that the quaternary assembly of MmPDE5A1 is a mixture of dimers and tetramers, while MmPDE5A2 and MmPDE5A3 only assembled as dimers. We showed that the N-terminal peptide is responsible for the tetramer assembly of MmPDE5A1, while that of the MmPDE5A2 is responsible for its mitochondrial localization. Overexpression of the three isoforms alters at different levels the cAMP/cGMP equilibrium as well as the NAD(P)⁺/NAD(P)H balance and induces a metabolic switch from oxidative to fermentative. In particular, the mitochondrial localization of MmPDE5A2 unveiled the existence of a cAMP-cGMP signaling cascade in this organelle, for which we propose a metabolic model that could explain the role of PDE5 in some cardiomyopathies and some of the side effects of its inhibitors.     

Inhibition of Phosphodiesterase-4 in Psoriatic Arthritis and Inflammatory Bowel Diseases

Phosphodiesterases (PDEs) are a heterogeneous superfamily of enzymes which catalyze the degradation of the intracellular second messengers cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP). Among PDEs, PDE4 is the most widely studied and characterized isoenzyme. PDE4 blocking can lead to increased levels of intracellular cAMP, which results in down-regulation of inflammatory responses by reducing the expression of tumor necrosis factor (TNF), interleukin (IL)-23, IL-17, interferon-γ, while increasing regulatory cytokines, such as IL-10. Therefore, PDE4 has been explored as a therapeutic target for the treatment of different chronic inflammatory conditions such as psoriatic arthritis (PsA) and inflammatory bowel disease (IBD). PsA shares clinical, genetic, and pathogenic features with IBD such as ulcerative colitis (UC) and Crohn’s disease (CD), and enteropathic spondyloarthritis (eSpA) represent a frequent clinical evidence of the overlap between gut and joint diseases. Current therapeutic options in PsA patients and underlying UC are limited to synthetic immunosuppressants and anti-TNF. Apremilast is an oral PDE4 inhibitor approved for the treatment of active PsA patients with inadequate response to synthetic immunosuppressants. The efficacy and a good safety profile observed in randomized clinical trials with apremilast in PsA patients have been confirmed by few studies in a real-life scenario. In addition, apremilast led to significant improvement in clinical and endoscopic features in UC patients in a phase II RCT. By now there are no available data regarding its role in eSpA patients. In view of the above, the use of apremilast in eSpA patients is a route that deserves to be deepened.     

Enhanced cGMP Interactor Rap Guanine Exchange Factor 4 (EPAC2) Expression and Activity in Degenerating Photoreceptors: A Neuroprotective Response?

The disease retinitis pigmentosa (RP) leads to photoreceptor degeneration by a yet undefined mechanism(s). In several RP mouse models (i.e., rd mice), a high cyclic GMP (cGMP) level within photoreceptors is detected, suggesting that cGMP plays a role in degeneration. The rap guanine exchange factor 4 (EPAC2) is activated by cyclic AMP (cAMP) and is an accepted cGMP-interacting protein. It is unclear whether and how cGMP interacts with EPAC2 in degenerating photoreceptors; we therefore investigated EPAC2 expression and interactions with cGMP and cAMP in retinas of the rd1 and rd10 models for retinal degeneration. EPAC2 expression in the photoreceptor layer increased significantly during rd1 and rd10 degeneration, and an increase in EPAC2 interactions with cGMP but not cAMP in the rd1 was also seen via a proximity ligation assay on histological sections. Retinal explant cultures revealed that pharmacological inhibition of the EPAC2 activity reduced the photoreceptor layer thickness in the rd10 retina, suggesting that EPAC2 inhibition promotes degeneration. Taken together, our results support the hypothesis that high degeneration-related cGMP leads to increased EPAC2 and cGMP interactions, inhibiting EPAC2. By inference, EPAC2 could have neuroprotective capacities that may be exploited in the future.     

Cyclic nucleotide signalling in kidney fibrosis

Kidney fibrosis is an important factor for the progression of kidney diseases, e.g., diabetes mellitus induced kidney failure, glomerulosclerosis and nephritis resulting in chronic kidney disease or end-stage renal disease. Cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were implicated to suppress several of the above mentioned renal diseases. In this review article, identified effects and mechanisms of cGMP and cAMP regarding renal fibrosis are summarized. These mechanisms include several signalling pathways of nitric oxide/ANP/guanylyl cyclases/cGMP-dependent protein kinase and cAMP/Epac/adenylyl cyclases/cAMP-dependent protein kinase. Furthermore, diverse possible drugs activating these pathways are discussed. From these diverse mechanisms it is expected that new pharmacological treatments will evolve for the therapy or even prevention of kidney failure.     

A Real-Time,Plate-Based BRET Assay for Detection of cGMP in Primary Cells

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.     

Compartmentalization Role of A-Kinase Anchoring Proteins (AKAPs) in Mediating Protein Kinase A (PKA) Signaling and Cardiomyocyte Hypertrophy

The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure–function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.     

Expression Pattern of Purinergic Signaling Components in Colorectal Cancer Cells and Differential Cellular Outcomes Induced by Extracellular ATP and Adenosine

The purine nucleotide adenosine triphosphate (ATP) is known for its fundamental role in cellular bioenergetics. However, in the last decades, different works have described emerging functions for ATP, such as that of a danger signaling molecule acting in the extracellular space on both tumor and stromal compartments. Beside its role in immune cell signaling, several studies have shown that high concentrations of extracellular ATP can directly or indirectly act on cancer cells. Accordingly, it has been reported that purinergic receptors are widely expressed in tumor cells. However, their expression pattern is often associated with contradictory cellular outcomes. In this work, we first investigated gene expression profiles through “RNA-Sequencing” (RNA Seq) technology in four colorectal cancer (CRC) cell lines (HT29, LS513, LS174T, HCT116). Our results demonstrate that CRC cells mostly express the A2B, P2X4, P2Y1, P2Y2 and P2Y11 purinergic receptors. Among these, the P2Y1 and P2Y2 coding genes are markedly overexpressed in all CRC cells compared to the HCEC-1CT normal-like colonic cells. We then explored the cellular outcomes induced by extracellular ATP and adenosine. Our results show that in terms of cell death induction extracellular ATP is consistently more active than adenosine against CRC, while neither compound affected normal-like colonic cell survival. Intriguingly, while for the P2Y2 receptor pharmacological inhibition completely abolished the rise in cytoplasmic Ca²⁺ observed after ATP exposure in all CRC cell lines, Ca²⁺ mobilization only impacted the cellular outcome for HT29. In contrast, non-selective phosphodiesterase inhibition completely abolished the effects of extracellular ATP on CRC cells, suggesting that cAMP and/or cGMP levels might determine cellular outcome. Altogether, our study provides novel insights into the characterization of purinergic signaling in CRC.     

BdGUCD1 and Cyclic GMP Are Required for Responses of Brachypodium distachyon to Fusarium pseudograminearum in the Mechanism Involving Jasmonate

Guanosine 3′,5′-cyclic monophosphate (cGMP) is an important signaling molecule in plants. cGMP and guanylyl cyclases (GCs), enzymes that catalyze the synthesis of cGMP from GTP, are involved in several physiological processes and responses to environmental factors, including pathogen infections. Using in vitro analysis, we demonstrated that recombinant BdGUCD1 is a protein with high guanylyl cyclase activity and lower adenylyl cyclase activity. In Brachypodium distachyon, infection by Fusarium pseudograminearum leads to changes in BdGUCD1 mRNA levels, as well as differences in endogenous cGMP levels. These observed changes may be related to alarm reactions induced by pathogen infection. As fluctuations in stress phytohormones after infection have been previously described, we performed experiments to determine the relationship between cyclic nucleotides and phytohormones. The results revealed that inhibition of cellular cGMP changes disrupts stress phytohormone content and responses to pathogen. The observations made here allow us to conclude that cGMP is an important element involved in the processes triggered as a result of infection and changes in its levels affect jasmonic acid. Therefore, stimuli-induced transient elevation of cGMP in plants may play beneficial roles in priming an optimized response, likely by triggering the mechanisms of feedback control.     

Examination of Intracellular GPCR-Mediated Signaling with High Temporal Resolution

The GTP-binding protein-coupled receptors (GPCRs) play important roles in physiology and neuronal signaling. More than a thousand genes, excluding the olfactory receptors, have been identified that encode these integral membrane proteins. Their pharmacological and functional properties make them fascinating targets for drug development, since various disease states can be treated and overcome by pharmacologically addressing these receptors and/or their downstream interacting partners. The activation of the GPCRs typically causes transient changes in the intracellular second messenger concentrations as well as in membrane conductance. In contrast to ion channel-mediated electrical signaling which results in spontaneous cellular responses, the GPCR-mediated metabotropic signals operate at a different time scale. Here we have studied the kinetics of two common GPCR-induced signaling pathways: (a) Ca²⁺ release from intracellular stores and (b) cyclic adenosine monophosphate (cAMP) production. The latter was monitored via the activation of cyclic nucleotide-gated (CNG) ion channels causing Ca²⁺ influx into the cell. Genetically modified and stably transfected cell lines were established and used in stopped-flow experiments to uncover the individual steps of the reaction cascades. Using two homologous biogenic amine receptors, either coupling to G_o/q or G_s proteins, allowed us to determine the time between receptor activation and signal output. With ~350 ms, the release of Ca²⁺ from intracellular stores was much faster than cAMP-mediated Ca²⁺ entry through CNG channels (~6 s). The measurements with caged compounds suggest that this difference is due to turnover numbers of the GPCR downstream effectors rather than the different reaction cascades, per se.     

Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3

Interleukin-1 receptor-associated kinase-3 (IRAK3) is a critical checkpoint molecule of inflammatory responses in the innate immune system. The pseudokinase domain of IRAK3 contains a guanylate cyclase (GC) centre that generates small amounts of cyclic guanosine monophosphate (cGMP) associated with IRAK3 functions in inflammation. However, the mechanisms of IRAK3 actions are poorly understood. The effects of low cGMP levels on inflammation are unknown, therefore a dose–response effect of cGMP on inflammatory markers was assessed in THP-1 monocytes challenged with lipopolysaccharide (LPS). Sub-nanomolar concentrations of membrane permeable 8-Br-cGMP reduced LPS-induced NFκB activity, IL-6 and TNF-α cytokine levels. Pharmacologically upregulating cellular cGMP levels using a nitric oxide donor reduced cytokine secretion. Downregulating cellular cGMP using a soluble GC inhibitor increased cytokine levels. Knocking down IRAK3 in THP-1 cells revealed that unlike the wild type cells, 8-Br-cGMP did not suppress inflammatory responses. Complementation of IRAK3 knockdown cells with wild type IRAK3 suppressed cytokine production while complementation with an IRAK3 mutant at GC centre only partially restored this function. Together these findings indicate low levels of cGMP form a critical component in suppressing cytokine production and in mediating IRAK3 action, and this may be via a cGMP enriched nanodomain formed by IRAK3 itself.     

Mitochondrial a Kinase Anchor Proteins in Cardiovascular Health and Disease: A Review Article on Behalf of the Working Group on Cellular and Molecular Biology of the Heart of the Italian Society of Cardiology

Second messenger cyclic adenosine monophosphate (cAMP) has been found to regulate multiple mitochondrial functions, including respiration, dynamics, reactive oxygen species production, cell survival and death through the activation of cAMP-dependent protein kinase A (PKA) and other effectors. Several members of the large family of A kinase anchor proteins (AKAPs) have been previously shown to locally amplify cAMP/PKA signaling to mitochondria, promoting the assembly of signalosomes, regulating multiple cardiac functions under both physiological and pathological conditions. In this review, we will discuss roles and regulation of major mitochondria-targeted AKAPs, along with opportunities and challenges to modulate their functions for translational purposes in the cardiovascular system.     

Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP–Epac–CREB signaling pathway to upregulate FGF21 expression in hepatocytes.     

The Complex Story of Plant Cyclic Nucleotide-Gated Channels

Plant cyclic nucleotide-gated channels (CNGCs) are tetrameric cation channels which may be activated by the cyclic nucleotides (cNMPs) adenosine 3′,5′-cyclic monophosphate (cAMP) and guanosine 3′,5′-cyclic monophosphate (cGMP). The genome of Arabidopsis thaliana encodes 20 CNGC subunits associated with aspects of development, stress response and immunity. Recently, it has been demonstrated that CNGC subunits form heterotetrameric complexes which behave differently from the homotetramers produced by their constituent subunits. These findings have widespread implications for future signalling research and may help explain how specificity can be achieved by CNGCs that are known to act in disparate pathways. Regulation of complex formation may involve cyclic nucleotide-gated channel-like proteins.     

Endoplasmic Reticulum‐Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling

In eukaryotic cells, ultimate specificity in activation and action—for example, by means of second messengers—of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca²⁺) and cyclic adenosine monophosphate (cAMP), regulate multiple—sometimes opposite—cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca²⁺ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca²⁺ channels at ER–PM contact sites. Within the MCS, Ca²⁺ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.     

Distinct Roles of Soluble and Transmembrane Adenylyl Cyclases in the Regulation of Flagellar Motility in Ciona Sperm

Adenylyl cyclase (AC) is a key enzyme that synthesizes cyclic AMP (cAMP) at the onset of the signaling pathway to activate sperm motility. Here, we showed that both transmembrane AC (tmAC) and soluble AC (sAC) are distinctly involved in the regulation of sperm motility in the ascidian Ciona intestinalis. A tmAC inhibitor blocked both cAMP synthesis and the activation of sperm motility induced by the egg factor sperm activating and attracting factor (SAAF), as well as those induced by theophylline, an inhibitor of phoshodiesterase. It also significantly inhibited cAMP-dependent phosphorylation of a set of proteins at motility activation. On the other hand, a sAC inhibitor does not affect on SAAF-induced transient increase of cAMP, motility activation or protein phosphorylation, but it reduced swimming velocity to half in theophylline-induced sperm. A sAC inhibitor KH-7 induced circular swimming trajectory with smaller diameter and significantly suppressed chemotaxis of sperm to SAAF. These results suggest that tmAC is involved in the basic mechanism for motility activation through cAMP-dependent protein phosphorylation, whereas sAC plays distinct roles in increase of flagellar beat frequency and in the Ca²⁺-dependent chemotactic movement of sperm.     

Dihydroaustrasulfone Alcohol (WA-25) Impedes Macrophage Foam Cell Formation by Regulating the Transforming Growth Factor-β1 Pathway

Atherosclerosis is considered an inflammatory disease. However, clinically used anti-atherosclerotic drugs, such as simvastatin, have many side effects. Recently, several unique marine compounds have been isolated that possess a variety of bioactivities. In a previous study, we found a synthetic precursor of the marine compound (austrasulfone), which is dihydroaustrasulfone alcohol (WA-25), has anti-atherosclerotic effects in vivo. However, the detailed mechanisms remain unclear. Therefore, to clarify the mechanisms through which WA-25 exerts anti-atherosclerotic activity, we used RAW 264.7 macrophages as an in vitro model to evaluate the effects of WA-25. In lipopolysaccharide (LPS)-stimulated RAW 264.7 cells, WA-25 significantly inhibited expression of the pro-inflammatory proteins, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In contrast, simvastatin increased the COX-2 expression compared to WA-25. In addition, WA-25 impedes foam cell formation and up-regulated the lysosomal and cyclic adenosine monophosphate (cAMP) signaling pathway. We also observed that transforming growth factor β1 (TGF-β1) was up-regulated by WA-25 and simvastatin in LPS-induced RAW 264.7 cells, and the promising anti-atherosclerosis effects of WA-25 were disrupted by blockade of TGF-β1 signaling. Besides, WA-25 might act through increasing lipolysis than through alteration of lipid export. Taken together, these data demonstrate that WA-25 may have potential as an anti-atherosclerotic drug with anti-inflammatory effects.