PHOSPHODIESTERASE-4 INHIBITION REDUCES PROTEOLYSIS AND ATROGENES EXPRESSION IN RAT SKELETAL MUSCLES

Phosphodiesterase (PDE) inhibition reduces skeletal muscle atrophy, but the underlying molecular mechanism remains unclear. We used microdialysis to investigate the effects of different PDE inhibitors on interstitial tyrosine concentration as well as proteolytic activity and atrogenes expression in isolated rat muscle. Rolipram, a PDE-4-selective inhibitor, reduced the interstitial tyrosine concentration and rates of muscle protein degradation. The rolipram-induced muscle cAMP increase was accompanied by a decrease in ubiquitin proteasome system (UPS) activity and atrogin-1 mRNA, a ubiquitin-ligase involved in muscle atrophy. This effect was not associated with Akt phosphorylation but was partially blocked by a protein kinase A inhibitor. Fasting increased atrogin-1, MuRF-1 and LC3b expression, and these effects were markedly suppressed by rolipram. Our data suggest that activation of cAMP signaling by PDE-4 blockade leads to inhibition of UPS activity and atrogenes expression independently of Akt. These findings are important for identifying novel approaches to attenuate muscle atrophy. Muscle Nerve 44: 371-381, 2011

Comparison of the modes of action of Apremilast and Roflumilast

The phosphodiesterase 4 (PDE4) family are cAMP specific phosphodiesterases that play an important role in the inflammatory response and is the major PDE type found in inflammatory cells. A significant number of PDE4 specific inhibitors have been developed and are currently being investigated for use as therapeutic agents. Apremilast, a small molecule inhibitor of PDE 4 is in development for chronic inflammatory disorders and has shown promise for the treatment of psoriasis, psoriatic arthritis as well as other inflammatory diseases. It has been found to be safe and well tolerated in humans and in March 2014 it was approved by the US food and drug administration for the treatment of adult patients with active psoriatic arthritis. The only other PDE4 inhibitor on the market is Roflumilast and it is used for treatment of respiratory disease. Roflumilast is approved in the EU for the treatment of COPD and was recently approved in the US for treatment to reduce the risk of COPD exacerbations. Roflumilast is also a selective PDE4 inhibitor, administered as an oral tablet once daily, and is thought to act by increasing cAMP within lung cells. As both (Apremilast and Roflumilast) compounds selectively inhibit PDE4 but are targeted at different diseases, there is a need for a clear understanding of their mechanism of action (MOA). Differences and similarity of MOA should be defined for the purposes of labelling, for communication to the scientific community, physicians, and patients, and for an extension of utility to other diseases and therapeutic areas. In order to obtain a complete comparative picture of the MOA of both inhibitors, additional molecular and cellular biology studies are required to more fully elucidate the signalling mediators downstream of PDE4 inhibition which result in alterations in pro- and anti-inflammatory gene expression. My studies were conducted to directly compare Apremilast with Roflumilast, in order to substantiate the differences observed in the molecular and cellular effects of these compounds, and to search for other possible differentiating effects. Therefore the main aim of this thesis was to utilise cutting-edge biochemical techniques to discover whether Apremilast and Roflumilast work with different modes of action. In the first part of my thesis I used novel genetically encoded FRET based cAMP sensors targeted to different intracellular compartments, in order to monitor cAMP levels within specific microdomains of cells as a consequence of challenge with Apremilast and Roflumilast, which revealed that Apremilast and Roflumilast do regulate different pools of cAMP in cells. In the second part of my thesis I focussed on assessing whether Apremilast and Roflumilast cause differential effects on the PKA phosphorylation state of proteins in cells. I used various biochemical techniques (Western blotting, Substrate kinase arrays and Reverse Phase Protein array and found that Apremilast and Roflumilast do lead to differential PKA substrate phosphorylation. For example I found that Apremilast increases the phosphorylation of Ribosomal Protein S6 at Ser240/244 and Fyn Y530 in the S6 Ribosomal pathway of Rheumatoid Arthritis Synovial fibroblast and HEK293 cells, whereas Roflumilast does not. This data suggests that Apremilast has distinct biological effects from that of Roflumilast and could represent a new therapeutic role for Apremilast in other diseases. In the final part of my thesis, Phage display technology was employed in order to identify any novel binding motifs that associate with PDE4 and to identify sequences that were differentially regulated by the inhibitors in an attempt to find binding motifs that may exist in previously characterised signalling proteins. Petide array technology was then used to confirm binding of specific peptide sequences or motifs. Results showed that Apremilast and Roflumilast can either enhance or decrease the binding of PDE4A4 to specific peptide sequences or motifs that are found in a variety of proteins in the human proteome, most interestingly Ubiquitin-related proteins. The data from this chapter is preliminary but may be used in the discovery of novel binding partners for PDE4 or to provide a new role for PDE inhibition in disease. Therefore the work in this thesis provides a unique snapshot of the complexity of the cAMP signalling system and is the first to directly compare action of the two approved PDE4 inhibitors in a detailed way.

Effects Of Pde Type 5 Inhibitors On Left Ventricular Diastolic Dysfunction In Resistant Hypertension.

Resistant hypertension (RHTN) is a multifactorial disease characterized by blood pressure (BP) levels above goal (140/90 mmHg) in spite of the concurrent use of three or more antihypertensive drugs of different classes. Moreover, it is well known that RHTN subjects have high prevalence of left ventricular diastolic dysfunction (LVDD), which leads to increased risk of heart failure progression. This review gathers data from studies evaluating the effects of phosphodiesterase-5 (PDE-5) inhibitors (administration of acute sildenafil and short-term tadalafil) on diastolic function, biochemical and hemodynamic parameters in patients with RHTN. Acute study with sildenafil treatment found that inhibition of PDE-5 improved hemodynamic parameters and diastolic relaxation. In addition, short-term study with the use of tadalafil demonstrated improvement of LVDD, cGMP and BNP-32 levels, regardless of BP reduction. No endothelial function changes were observed in the studies. The findings of acute and short-term studies revealed potential therapeutic effects of IPDE-5 drugs on LVDD in RHTN patients.A Hipertensão arterial resistente (HAR) é uma doença multifatorial caracterizada por níveis pressóricos acima das metas (140/90 mmHg), a despeito de tratamento farmacológico otimizado de 3 ou mais fármacos anti-hipertensivos de diferentes classes. Pacientes diagnosticados como hipertensos resistentes apresentam alta prevalência de disfunção diastólica do ventrículo esquerdo (DDVE) que proporciona risco aumentado para insuficiência cardíaca. Esta revisão reúne dados de estudos prévios avaliando os efeitos dos inibidores de fosfodiesterase-5 (PDE-5) (administração aguda de sildenafil e de curto prazo de tadalafil) na função diastólica e nos parâmetros bioquímicos e hemodinâmicos em pacientes com HAR. O estudo agudo com sildenafil demonstrou que a inibição da PDE-5 melhorou os parâmetros hemodinâmicos e de relaxamento diastólico. Além disso, o estudo curto prazo com o uso de tadalafil revelou melhora da DDVE e dos níveis de GMPc e BNP-32, independente de redução de pressão arterial. A função endotelial não apresentou alteração com ambos os tratamentos. Os resultados dos estudos agudo e de curto prazo sugerem efeitos terapêuticos potenciais dos fármacos inibidores da PDE-5 na disfunção diastólica em pacientes com HAR.

Effects of PDE type 5 inhibitors on Left Ventricular Diastolic Dysfunction in Resistant Hypertension

Resistant hypertension (RHTN) is a multifactorial disease characterized by blood pressure (BP) levels above goal (140/90 mmHg) in spite of the concurrent use of three or more antihypertensive drugs of different classes. Moreover, it is well known that RHTN subjects have high prevalence of left ventricular diastolic dysfunction (LVDD), which leads to increased risk of heart failure progression. This review gathers data from studies evaluating the effects of phosphodiesterase-5 (PDE-5) inhibitors (administration of acute sildenafil and short-term tadalafil) on diastolic function, biochemical and hemodynamic parameters in patients with RHTN. Acute study with sildenafil treatment found that inhibition of PDE-5 improved hemodynamic parameters and diastolic relaxation. In addition, short-term study with the use of tadalafil demonstrated improvement of LVDD, cGMP and BNP-32 levels, regardless of BP reduction. No endothelial function changes were observed in the studies. The findings of acute and short-term studies revealed potential therapeutic effects of IPDE-5 drugs on LVDD in RHTN patients.

Gene Expression in Bovine Oocytes and Cumulus Cells After Meiotic Inhibition with the Cyclin-Dependent Kinase Inhibitor Butyrolactone I

Contents The aim of this study was to determine the effect of temporary inhibition of meiosis using the cyclin-dependent kinase inhibitor butyrolactone I (BLI) on gene expression in bovine oocytes and cumulus cells. Immature bovine cumulusoocyte complexes (COCs) were assigned to groups: (i) Control COCs collected immediately after recovery from the ovary or (ii) after in vitro maturation (IVM) for 24 h, (iii) Inhibited COCs collected 24 h after incubation with 100 mu m BLI or (iv) after meiotic inhibition for 24 h followed by IVM for a further 22 h. For mRNA relative abundance analysis, pools of 10 denuded oocytes and respective cumulus cells were collected. Transcripts related to cell cycle regulation and oocyte competence were evaluated in oocytes and cumulus cells by quantitative real-time PCR (qPCR). Most of the examined transcripts were downregulated (p < 0.05) after IVM in control and inhibited oocytes (19 of 35). Nine transcripts remained stable (p > 0.05) after IVM in control oocytes; only INHBA did not show this pattern in inhibited oocytes. Seven genes were upregulated after IVM in control oocytes (p < 0.05), and only PLAT, RBP1 and INHBB were not upregulated in inhibited oocytes after IVM. In cumulus cells, six genes were upregulated (p < 0.05) after IVM and eight were downregulated (p < 0.05). Cells from inhibited oocytes showed the same pattern of expression regarding maturation profile, but were affected by the temporary meiosis inhibition of the oocyte when the same maturation stages were compared between inhibited and control groups. In conclusion, changes in transcript abundance in oocytes and cumulus cells during maturation in vitro were mostly mirrored after meiotic inhibition followed by maturation.

Mechanism of photoreceptor cGMP phosphodiesterase inhibition by its gamma-subunits.

cGMP phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells that regulates the level of the second messenger, cGMP. PDE consists of catalytic alpha and beta subunits (Palpha and Pbeta) and two inhibitory gamma subunits (Pgamma) that block PDE activity in the dark. The major inhibitory region has been localized to the C terminus of Pgamma. The last C-terminal residues -IleIle form an important hydrophobic domain critical for the inhibition of PDE activity. In this study, mutants of Pgamma were designed for cross-linking experiments to identify regions on Palpha and Pbeta subunits that bind to the Pgamma C terminus. In one of the mutants, the cysteine at position 68 was substituted with serine, and the last four C-terminal residues of Pgamma were replaced with a single cysteine. This mutant, Pgamma83Cys, was labeled with photoprobe 4-(N-maleimido) benzophenone (MBP) at the cysteine residue. The labeled Pgamma83CysMBP mutant was a more potent inhibitor of PDE activity than the unlabeled mutant, indicating that the hydrophobic MBP probe mimics the Pgamma hydrophobic C terminus. A specific, high-yield cross-linking of up to 70% was achieved between the Pgamma83CysMBP and PDE catalytic subunits. Palpha and the N-terminally truncated Pbeta (lacking 147 aa residues) cross-linked to Pgamma83CysMBP with the same efficiency. Using mass spectrometric analysis of tryptic fragments from the cross-linked PDE, we identified the site of cross-linking to aa residues 751-763 of Palpha. The corresponding region of Pbeta, Pbeta-749-761, also may bind to the Pgamma C terminus. Our data suggest that Pgamma blocks PDE activity through the binding to the catalytic site of PDE, near the NKXD motif, a consensus sequence for interaction with the guanine ring of cGMP.

Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Fatty Acid Synthase Inhibitors Induce Apoptosis In Non-tumorigenic Melan-a Cells Associated With Inhibition Of Mitochondrial Respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.

Effects Of Partial Inhibition Of Respiratory Complex I On H2o 2 Production By Isolated Brain Mitochondria In Different Respiratory States.

The aim of this work was to characterize the effects of partial inhibition of respiratory complex I by rotenone on H2O2 production by isolated rat brain mitochondria in different respiratory states. Flow cytometric analysis of membrane potential in isolated mitochondria indicated that rotenone leads to uniform respiratory inhibition when added to a suspension of mitochondria. When mitochondria were incubated in the presence of a low concentration of rotenone (10 nm) and NADH-linked substrates, oxygen consumption was reduced from 45.9 ± 1.0 to 26.4 ± 2.6 nmol O2 mg(-1) min(-1) and from 7.8 ± 0.3 to 6.3 ± 0.3 nmol O2 mg(-1) min(-1) in respiratory states 3 (ADP-stimulated respiration) and 4 (resting respiration), respectively. Under these conditions, mitochondrial H2O2 production was stimulated from 12.2 ± 1.1 to 21.0 ± 1.2 pmol H2O2 mg(-1) min(-1) and 56.5 ± 4.7 to 95.0 ± 11.1 pmol H2O2 mg(-1) min(-1) in respiratory states 3 and 4, respectively. Similar results were observed when comparing mitochondrial preparations enriched with synaptic or nonsynaptic mitochondria or when 1-methyl-4-phenylpyridinium ion (MPP(+)) was used as a respiratory complex I inhibitor. Rotenone-stimulated H2O2 production in respiratory states 3 and 4 was associated with a high reduction state of endogenous nicotinamide nucleotides. In succinate-supported mitochondrial respiration, where most of the mitochondrial H2O2 production relies on electron backflow from complex II to complex I, low rotenone concentrations inhibited H2O2 production. Rotenone had no effect on mitochondrial elimination of micromolar concentrations of H2O2. The present results support the conclusion that partial complex I inhibition may result in mitochondrial energy crisis and oxidative stress, the former being predominant under oxidative phosphorylation and the latter under resting respiration conditions.

Tadalafil-induced Improvement In Left Ventricular Diastolic Function In Resistant Hypertension.

Left ventricular hypertrophy and diastolic dysfunction (LVDD) remain highly frequent markers of cardiac damage and risk of progression to symptomatic heart failure, especially in resistant hypertension (RHTN). We have previously demonstrated that administration of sildenafil in hypertensive rats improves LVDD, restoring phosphodiesterase type 5 (PDE-5) inhibition in cardiac myocytes. We hypothesized that the long-acting PDE-5 inhibitor tadalafil may be clinically useful in improving LVDD in RHTN independently of blood pressure (BP) reduction. A single blinded, placebo-controlled, crossover study enrolled 19 patients with both RHTN and LVDD. Firstly, subjects received tadalafil (20 mg) for 14 days and after a 2-week washout period, they received placebo orally for 14 days. Patients were evaluated by office BP and ambulatory BP monitoring (ABPM), endothelial function (FMD), echocardiography, plasma brain natriuretic peptide (BNP-32), cyclic guanosine monophosphate (cGMP) and nitrite levels. No significant differences were detected in BP measurements. Remarkably, at least four echocardiographic parameters related with diastolic function improved accompanied by decrease in BNP-32 in tadalafil use. Although increasing cGMP, tadalafil did not change endothelial function or nitrites. There were no changes in those parameters after placebo. The current findings suggest that tadalafil improves LV relaxation through direct effects PDE-5-mediated in the cardiomyocytes with potential benefit as an adjunct to treat symptomatic subjects with LVDD such as RHTN patients.

Inhibition Of Tumor Necrosis Factor-alpha And Cyclooxigenase-2 By Isatin: A Molecular Mechanism Of Protection Against Tnbs-induced Colitis In Rats.

Isatin, an indole alkaloid has been shown to have anti-microbial, anti-tumor and anti-inflammatory effects. Due to its findings, we evaluated whether this alkaloid would have any effect on TNBS-induced colitis. Animals (male Unib:WH rats, aged 8 weeks old) were induced colitis through a rectal administration of 2,4,6-trinitrobenzene sulphonic acid using a catheter inserted 8 cm into the rectum of the animals. The rats were divided into two major groups: non-colitic and colitic. The colitic group was sub-divided into 6 groups (10 animals per group): colitic non-treated, Isatin 3; 6; 12.5; 18.75 and 25 mg/kg. Our main results showed that the oral treatment with Isatin 6 and 25 mg/kg were capable of avoiding the increase in TNF-α, COX-2 and PGE₂ levels when compared to the colitic non-treated group. Interestingly, the same doses (6 and 25 mg/kg) were also capable of preventing the decrease in IL-10 levels comparing with the colitic non-treated group. The levels of MPO, (an indirect indicator of neutrophil presence), were also maintained lower than those of the colitic non-treated group. Isatin also prevented the decrease of SOD activity and increase of GSH-Px and GSH-Rd activity as well as the depletion of GSH levels. In conclusion, both pre-treatments (6 and 25 mg/kg) were capable of protecting the gut mucosa against the injury caused by TNBS, through the combination of antioxidant and anti-inflammatory properties, which, together, showed a protective activity of the indole alkaloid Isatin.

Reduced cortical renal GLUT1 expression induced by angiotensin-converting enzyme inhibition in diabetic spontaneously hypertensive rats

Diabetes in spontaneously hypertensive rats is associated with cortical renal GLUT1 and GLUT2 overexpression. Our objective was to evaluate the effect of the angiotensin-converting enzyme blockade on cortical renal GLUT1 and GLUT2 expression, urinary albumin and urinary TGF-β1. Streptozotocin, 50 mg/kg, or citrate buffer (N = 16) was administered as a single injection into the tail vein in adult spontaneously hypertensive rats (~260 g). Thirty days later, these diabetic spontaneously hypertensive rats received ramipril by gavage: 0.01 mg·kg-1·day-1 (D0.01, N = 14), 1 mg·kg-1·day-1 (D1, N = 9) or water (D, N = 11) for 15 days. Albumin and TGF-β1 (24-h urine), direct arterial pressure, renal tissue angiotensin-converting enzyme activity (fluorometric assay), and GLUT1 and GLUT2 protein levels (Western blot, renal cortex) were determined. Glycemia and glycosuria were higher (P < 0.05) in the diabetic rats compared with controls, but similar between the diabetic groups. Diabetes in spontaneously hypertensive rats lowered renal tissue angiotensin-converting enzyme activity (40%), which was reduced further when higher ramipril doses were used. Diabetes associated with hypertension raised GLUT1 by 28% (P < 0.0001) and GLUT2 by 76% (P = 0.01), and both doses of ramipril equally reduced cortical GLUT1 (D vs D1 and vs D0.01, P ≤ 0.001). GLUT2 levels were reduced in D0.01 (P < 0.05 vs D). Diabetes increased urinary albumin and TGF-β1 urinary excretion, but the 15-day ramipril treatment (with either dose) did not reduce them. In conclusion, ramipril is effective in lowering renal tissue angiotensin-converting enzyme activity, as well as blocking cortical GLUT1 overexpression, which may be beneficial in arresting the development of diabetic nephropathy.

Proteasome Inhibition Represses Unfolded Protein Response and Nox4, Sensitizing Vascular Cells to Endoplasmic Reticulum Stress-Induced Death

Background: Endoplasmic reticulum (ER) stress has pathophysiological relevance in vascular diseases and merges with proteasome function. Proteasome inhibition induces cell stress and may have therapeutic implications. However, whether proteasome inhibition potentiates ER stress-induced apoptosis and the possible mechanisms involved in this process are unclear. Methodology/Principal Findings: Here we show that proteasome inhibition with MG132, per se at non-lethal levels, sensitized vascular smooth muscle cells to caspase-3 activation and cell death during ER stress induced by tunicamycin (Tn). This effect was accompanied by suppression of both proadaptive (KDEL chaperones) and proapoptotic (CHOP/GADD153) unfolded protein response markers, although, intriguingly, the splicing of XBP1 was markedly enhanced and sustained. In parallel, proteasome inhibition completely prevented ER stress-induced increase in NADPH oxidase activity, as well as increases in Nox4 isoform and protein disulfide isomerase mRNA expression. Increased Akt phosphorylation due to proteasome inhibition partially offset the proapoptotic effect of Tn or MG132. Although proteasome inhibition enhanced oxidative stress, reactive oxygen species scavenging had no net effect on sensitization to Tn or MG132-induced cell death. Conclusion/Relevance: These data indicate unfolded protein response-independent pathways whereby proteasome inhibition sensitizes vascular smooth muscle to ER stress-mediated cell death. This may be relevant to understand the therapeutic potential of such compounds in vascular disease associated with increased neointimal hyperplasia.

Leukotriene B(4) amplifies NF-kappa B activation in mouse macrophages by reducing SOCS1 inhibition of MyD88 expression

Activation of NF-kappa B and 5-lipoxygenase-mediated (5-LO-mediated) biosynthesis of the lipid mediator leukotriene B(4) (LTB(4)) are pivotal components of host defense and inflammatory responses. However, the role of LTB(4) in mediating innate immune responses elicited by specific TLR ligands and cytokines is unknown. Here we have shown that responses dependent on MyD88 (an adaptor protein that mediates signaling through all of the known TLRs, except TLR3, as well as IL-1 beta and IL-18) are reduced in mice lacking either 5-LO or the LTB(4) receptor BTL1, and that macrophages from these mice are impaired in MyD88-dependent activation of NF-kappa B. This macrophage defect was associated with lower basal and inducible expression of MyD88 and reflected impaired activation of STAT1 and overexpression of the STAT1 inhibitor SOCS1. Expression of MyD88 and responsiveness to the TLR4 ligand LPS were decreased by Stat1 siRNA silencing in WT macrophages and restored by Socs1 siRNA in 5-LO-deficient macrophages. These results uncover a pivotal role in macrophages for the GPCR BLT1 in regulating activation of NF-kappa B through Stat1-dependent expression of MyD88.