The Ocean's Pharmacy: Health Discoveries in Marine Algae.

Non-communicable diseases (NCDs) represent a global health challenge, constituting a major cause of mortality and disease burden in the 21st century. Addressing the prevention and management of NCDs is crucial for improving global public health, emphasizing the need for comprehensive strategies, early interventions, and innovative therapeutic approaches to mitigate their far-reaching consequences. Marine organisms, mainly algae, produce diverse marine natural products with significant therapeutic potential. Harnessing the largely untapped potential of algae could revolutionize drug development and contribute to combating NCDs, marking a crucial step toward natural and targeted therapeutic approaches. This review examines bioactive extracts, compounds, and commercial products derived from macro- and microalgae, exploring their protective properties against oxidative stress, inflammation, cardiovascular, gastrointestinal, metabolic diseases, and cancer across in vitro, cell-based, in vivo, and clinical studies. Most research focuses on macroalgae, demonstrating antioxidant, anti-inflammatory, cardioprotective, gut health modulation, metabolic health promotion, and anti-cancer effects. Microalgae products also exhibit anti-inflammatory, cardioprotective, and anti-cancer properties. Although studies mainly investigated extracts and fractions, isolated compounds from algae have also been explored. Notably, polysaccharides, phlorotannins, carotenoids, and terpenes emerge as prominent compounds, collectively representing 42.4% of the investigated compounds.

The therapeutic potential of sphingolipids for cardiovascular diseases.

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide and Inflammation plays a critical role in the development of CVD. Despite considerable progress in understanding the underlying mechanisms and various treatment options available, significant gaps in therapy necessitate the identification of novel therapeutic targets. Sphingolipids are a family of lipids that have gained attention in recent years as important players in CVDs and the inflammatory processes that underlie their development. As preclinical studies have shown that targeting sphingolipids can modulate inflammation and ameliorate CVDs, targeting sphingolipids has emerged as a promising therapeutic strategy. This review discusses the current understanding of sphingolipids' involvement in inflammation and cardiovascular diseases, the existing therapeutic approaches and gaps in therapy, and explores the potential of sphingolipids-based drugs as a future avenue for CVD treatment.

Riok1, A Novel Potential Target in MSI-High p53 Mutant Colorectal Cancer Cells.

The vulnerabilities of cancer cells constitute a promising strategy for drug therapeutics. This paper integrates proteomics, bioinformatics, and cell genotype together with in vitro cell proliferation assays to identify key biological processes and potential novel kinases that could account, at least in part, for the clinical differences observed in colorectal cancer (CRC) patients. This study started by focusing on CRC cell lines stratified by their microsatellite (MS) state and p53 genotype. It shows that cell-cycle checkpoint, metabolism of proteins and RNA, signal transduction, and WNT signaling processes are significantly more active in MSI-High p53-WT cell lines. Conversely, MSI-High cell lines with a mutant (Mut) p53 gene showed hyperactivation of cell signaling, DNA repair, and immune-system processes. Several kinases were linked to these phenotypes, from which RIOK1 was selected for additional exploration. We also included the KRAS genotype in our analysis. Our results showed that RIOK1's inhibition in CRC MSI-High cell lines was dependent on both the p53 and KRAS genotypes. Explicitly, Nintedanib showed relatively low cytotoxicity in MSI-High with both mutant p53 and KRAS (HCT-15) but no inhibition in p53 and KRAS WT (SW48) MSI-High cells. This trend was flipped in CRC MSI-High bearing opposite p53-KRAS genotypes (e.g., p53-Mut KRAS-WT or p53-WT KRAS-Mut), where observed cytotoxicity was more extensive compared to the p53-KRAS WT-WT or Mut-Mut cells, with HCT 116 (KRAS-Mut and p53-WT) being the most sensitive to RIOK1 inhibition. These results highlight the potential of our in silico computational approach to identify novel kinases in CRC sub-MSI-High populations as well as the importance of clinical genomics in determining drug potency.

Microalgae-Sustainable Source for Alternative Proteins and Functional Ingredients Promoting Gut and Liver Health.

Dietary proteins derived from animal sources, although containing well-balanced profiles of essential amino acids, have considerable environmental and adverse health effects associated with the intake of some animal protein-based products. Consuming foods based on animal proteins carries a higher risk of developing non-communicable diseases such as cancer, heart disease, non-alcoholic fatty liver disease (NAFLD), and inflammatory bowel disease (IBD). Moreover, dietary protein consumption is increasing due to population growth, posing a supply challenge. There is, therefore, growing interest in discovering novel alternative protein sources. In this context, microalgae have been recognized as strategic crops that can provide a sustainable source of protein. Compared to conventional high-protein crops, using microalgal biomass for protein production presents several advantages in food and feed in terms of productivity, sustainability, and nutritional value. Moreover, microalgae positively impact the environment by not exploiting land or causing water pollution. Many studies have revealed the potential of microalgae as an alternative protein source with the added value of positive effects on human health due to their anti-inflammatory, antioxidant, and anti-cancer properties. The main emphasis of this review is on the potential health-promoting applications of microalgae-based proteins, peptides, and bioactive substances for IBD and NAFLD.

Regulation of Cholesterol Metabolism by Phytochemicals Derived from Algae and Edible Mushrooms in Non-Alcoholic Fatty Liver Disease.

Cholesterol synthesis occurs in almost all cells, but mainly in hepatocytes in the liver. Cholesterol is garnering increasing attention for its central role in various metabolic diseases. In addition, cholesterol is one of the most essential elements for cells as both a structural source and a player participating in various metabolic pathways. Accurate regulation of cholesterol is necessary for the proper metabolism of fats in the body. Disturbances in cholesterol homeostasis have been linked to various metabolic diseases, such as hyperlipidemia and non-alcoholic fatty liver disease (NAFLD). For many years, the use of synthetic chemical drugs has been effective against many health conditions. Furthermore, from ancient to modern times, various plant-based drugs have been considered local medicines, playing important roles in human health. Phytochemicals are bioactive natural compounds that are derived from medicinal plants, fruit, vegetables, roots, leaves, and flowers and are used to treat a variety of diseases. They include flavonoids, carotenoids, polyphenols, polysaccharides, vitamins, and more. Many of these compounds have been proven to have antioxidant, anti-inflammatory, antiobesity and antihypercholesteremic activity. The multifaceted role of phytochemicals may provide health benefits to humans with regard to the treatment and control of cholesterol metabolism and the diseases associated with this disorder, such as NAFLD. In recent years, global environmental climate change, the COVID-19 pandemic, the current war in Europe, and other conflicts have threatened food security and human nutrition worldwide. This further emphasizes the urgent need for sustainable sources of functional phytochemicals to be included in the food industry and dietary habits. This review summarizes the latest findings on selected phytochemicals from sustainable sources-algae and edible mushrooms-that affect the synthesis and metabolism of cholesterol and improve or prevent NAFLD.

Flavonoids-Macromolecules Interactions in Human Diseases with Focus on Alzheimer, Atherosclerosis and Cancer.

Flavonoids, a class of polyphenols, consumed daily in our diet, are associated with a reduced risk for oxidative stress (OS)-related chronic diseases, such as cardiovascular disease, neurodegenerative diseases, cancer, and inflammation. The involvement of flavonoids with OS-related chronic diseases have been traditionally attributed to their antioxidant activity. However, evidence from recent studies indicate that flavonoids' beneficial impact may be assigned to their interaction with cellular macromolecules, rather than exerting a direct antioxidant effect. This review provides an overview of the recent evolving research on interactions between the flavonoids and lipoproteins, proteins, chromatin, DNA, and cell-signaling molecules that are involved in the OS-related chronic diseases; it focuses on the mechanisms by which flavonoids attenuate the development of the aforementioned chronic diseases via direct and indirect effects on gene expression and cellular functions. The current review summarizes data from the literature and from our recent research and then compares specific flavonoids' interactions with their targets, focusing on flavonoid structure-activity relationships. In addition, the various methods of evaluating flavonoid-protein and flavonoid-DNA interactions are presented. Our aim is to shed light on flavonoids action in the body, beyond their well-established, direct antioxidant activity, and to provide insights into the mechanisms by which these small molecules, consumed daily, influence cellular functions.

Photosynthetically Controlled Spirulina, but Not Solar Spirulina, Inhibits TNF-α Secretion: Potential Implications for COVID-19-Related Cytokine Storm Therapy.

An array of infections, including the novel coronavirus (SARS-CoV-2), trigger macrophage activation syndrome (MAS) and subsequently hypercytokinemia, commonly referred to as a cytokine storm (CS). It is postulated that CS is mainly responsible for critical COVID-19 cases, including acute respiratory distress syndrome (ARDS). Recognizing the therapeutic potential of Spirulina blue-green algae (Arthrospira platensis), in this in vitro stimulation study, LPS-activated macrophages and monocytes were treated with aqueous extracts of Spirulina, cultivated in either natural or controlled light conditions. We report that an extract of photosynthetically controlled Spirulina (LED Spirulina), at a concentration of 0.1 µg/mL, decreases macrophage and monocyte-induced TNF-α secretion levels by over 70% and 40%, respectively. We propose prompt in vivo studies in animal models and human subjects to determine the putative effectiveness of a natural, algae-based treatment for viral CS and ARDS, and explore the potential of a novel anti-TNF-α therapy.

Deletion or inhibition of SphK1 mitigates fulminant hepatic failure by suppressing TNFα-dependent inflammation and apoptosis.

Acute liver failure (ALF) causes severe liver dysfunction that can lead to multi-organ failure and death. Previous studies suggest that sphingosine kinase 1 (SphK1) protects against hepatocyte injury, yet not much is still known about its involvement in ALF. This study examines the role of SphK1 in D-galactosamine (GalN)/lipopolysaccharide (LPS)-induced ALF, which is a well-established experimental mouse model that mimics the fulminant hepatitis. Here we report that deletion of SphK1, but not SphK2, dramatically decreased GalN/LPS-induced liver damage, hepatic apoptosis, serum alanine aminotransferase levels, and mortality rate compared to wild-type mice. Whereas GalN/LPS treatment-induced hepatic activation of NF-κB and JNK in wild-type and SphK2-/- mice, these signaling pathways were reduced in SphK1-/- mice. Moreover, repression of ALF in SphK1-/- mice correlated with decreased expression of the pro-inflammatory cytokine TNFα. Adoptive transfer experiments indicated that SphK1 in bone marrow-derived infiltrating immune cells but not in host liver-resident cells, contribute to the development of ALF. Interestingly, LPS-induced TNFα production was drastically suppressed in SphK1-deleted macrophages, whereas IL-10 expression was markedly enhanced, suggesting a switch to the anti-inflammatory phenotype. Finally, treatment with a specific SphK1 inhibitor ameliorated inflammation and protected mice from ALF. Our findings suggest that SphK1 regulates TNFα secretion from macrophages and inhibition or deletion of SphK1 mitigated ALF. Thus, a potent inhibitor of SphK1 could potentially be a therapeutic agent for fulminant hepatitis.

Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) and the kinase that produces it have been implicated in inflammatory bowel diseases in mice and humans; however, little is known about the role of the 2 S1P-specific phosphohydrolase isoforms, SGPP1 and SGPP2, which catalyze dephosphorylation of S1P to sphingosine. To elucidate their functions, we generated specific knockout mice. Deletion of Sgpp2, which is mainly expressed in the gastrointestinal tract, significantly reduced dextran sodium sulfate (DSS)-induced colitis severity, whereas deletion of ubiquitously expressed Sgpp1 slightly worsened colitis. Moreover, Sgpp1 deletion enhanced expression of multifunctional proinflammatory cytokines, IL-6, TNF-α, and IL-1ß, activation of the transcription factor signal transducer and activator of transcription 3, and immune cell infiltration into the colon. Conversely, Sgpp2-null mice failed to mount a DSS-induced systemic inflammatory response. Of interest, Sgpp2 deficiency suppressed DSS-induced intestinal epithelial cell apoptosis and improved mucosal barrier integrity. Furthermore, down-regulation of Sgpp2 attenuated LPS-induced paracellular permeability in cultured cells and enhanced expression of the adherens junction protein E-cadherin. Finally, in patients with ulcerative colitis, SGPP2 expression was elevated in colitis tissues relative to that in uninvolved tissues. These results indicate that induction of SGPP2 expression contributes to the pathogenesis of colitis by promoting disruption of the mucosal barrier function. SGPP2 may represent a novel therapeutic target in inflammatory bowel disease.-Huang, W.-C., Liang, J., Nagahashi, M., Avni, D., Yamada, A., Maceyka, M., Wolen, A. R., Kordula, T., Milstien, S., Takabe, K., Oravecz, T., Spiegel, S. Sphingosine-1-phosphate phosphatase 2 promotes disruption of mucosal integrity, and contributes to ulcerative colitis in mice and humans.

Exogenous ceramide-1-phosphate (C1P) and phospho-ceramide analogue-1 (PCERA-1) regulate key macrophage activities via distinct receptors.

Inflammation is an ensemble of tightly regulated steps, in which macrophages play an essential role. Previous reports showed that the natural sphingolipid ceramide 1-phosphate (C1P) stimulates macrophages migration, while the synthetic C1P mimic, phospho-ceramide analogue-1 (PCERA-1), suppresses production of the key pro-inflammatory cytokine TNFα and amplifies production of the key anti-inflammatory cytokine IL-10 in LPS-stimulated macrophages, via one or more unidentified G-protein coupled receptors. We show that C1P stimulated RAW264.7 macrophages migration via the NFκB pathway and MCP-1 induction, while PCERA-1 neither mimicked nor antagonized these activities. Conversely, PCERA-1 synergistically elevated LPS-dependent IL-10 expression in RAW264.7 macrophages via the cAMP-PKA-CREB signaling pathway, while C1P neither mimicked nor antagonized these activities. Interestingly, both compounds have the capacity to additively inhibit TNFα secretion; PCERA-1, but not C1P, suppressed LPS-induced TNFα expression in macrophages in a CREB-dependent manner, while C1P, but not PCERA-1, directly inhibited recombinant TNFα converting enzyme (TACE). Finally, PCERA-1 failed to interfere with binding of C1P to either the cell surface receptor or to TACE. These results thus indicate that the natural sphingolipid C1P and its synthetic analog PCERA-1 bind and activate distinct receptors expressed in RAW264.7 macrophages. Identification of these receptors will be instrumental for elucidation of novel activities of extra-cellular sphingolipids, and may pave the way for the design of new sphingolipid mimics for the treatment of inflammatory diseases, and pathologies which depend on cell migration, as in metastatic tumors.

Aberrant ORM (yeast)-like protein isoform 3 (ORMDL3) expression dysregulates ceramide homeostasis in cells and ceramide exacerbates allergic asthma in mice.

BACKGROUND: Asthma, a chronic inflammatory condition defined by episodic shortness of breath with expiratory wheezing and cough, is a serious health concern affecting more than 250 million persons. Genome-wide association studies have identified ORM (yeast)-like protein isoform 3 (ORMDL3) as a gene associated with susceptibility to asthma. Although its yeast ortholog is a negative regulator of de novo ceramide biosynthesis, how ORMDL3 contributes to asthma pathogenesis is not known. OBJECTIVES: We sought to decipher the molecular mechanism for the pathologic functions of ORMDL3 in asthma and the relationship to its evolutionarily conserved role in regulation of ceramide homeostasis. METHODS: We determined the relationship between expression of ORMDL3 and ceramide in epithelial and inflammatory cells and in asthma pathogenesis in mice. RESULTS: Although small increases in ORMDL3 expression decrease ceramide levels, remarkably, higher expression in lung epithelial cells and macrophages in vitro and in vivo increased ceramide production, which promoted chronic inflammation, airway hyperresponsiveness, and mucus production during house dust mite-induced allergic asthma. Moreover, nasal administration of the immunosuppressant drug FTY720/fingolimod reduced ORMDL3 expression and ceramide levels and mitigated airway inflammation and hyperreactivity and mucus hypersecretion in house dust mite-challenged mice. CONCLUSIONS: Our findings demonstrate that overexpression of ORMDL3 regulates ceramide homeostasis in cells in a complex manner and suggest that local FTY720 administration might be a useful therapeutic intervention for the control of allergic asthma.

Nutritional aspects of honey bee-collected pollen and constraints on colony development in the eastern Mediterranean.

Pollen is the main protein and lipid source for honey bees (Apis mellifera), and nutritionally impoverished landscapes pose a threat to colony development. To determine colony nutritional demands, we analyzed a yearly cycle of bee-collected pollen from colonies in the field and compared it to colony worker production and honey bee body composition, for the first time in social insects. We monitored monthly bee production in ten colonies at each of seven sites throughout Israel, and trapped pollen bi-monthly in five additional colonies at each of four of these sites. Pollen mixtures from each sampling date and site were analyzed for weight, total protein, total fatty acids (FAs), and FA composition. Compared to more temperate climates, the eastern Mediterranean allows a relatively high yearly colony growth of ca. 300,000-400,000 bees. Colonies at higher elevation above sea level showed lower growth rates. Queen egg-laying rate did not seem to limit growth, as peaks in capped brood areas showed that queens lay a prolific 2000 eggs a day on average, with up to 3300 eggs in individual cases. Pollen uptake varied significantly among sites and seasons, with an overall annual mean total 16.8kg per colony, containing 7.14kg protein and 677g fat. Overall mean pollen protein content was high (39.8%), and mean total FA content was 3.8%. Production cost, as expressed by the amount of nutrient used per bee, was least variable for linoleic acid and protein, suggesting these as the best descriptive variables for total number of bees produced. Linolenic acid levels in pollen during the autumn were relatively low, and supplementing colonies with this essential FA may mitigate potential nutritional deficiency. The essentiality of linoleic and linolenic acids was consistent with these FAs' tendency to be present at higher levels in collected pollen than in the expected nutrients in bee bodies, demonstrating a well-developed adjustment between pollinator nutritional demands and the nutritional value of food offered by pollinated plants.

Role of sphingosine kinase 1 and sphingosine-1-phosphate in CD40 signaling and IgE class switching.

The tumor necrosis factor (TNF) receptor family member CD40 plays an essential role in the activation of antigen-presenting cells, B cell maturation, and immunoglobulin (Ig) class switching critical for adaptive immunity. Although the bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) and the kinase that produces it, sphingosine kinase 1 (SphK1), have long been implicated in the actions of TNF mediated by engagement of TNFR1, nothing is yet known of their role in CD40-mediated events. We have now found that ligation of CD40 activates and translocates SphK1 to the plasma membrane, leading to generation of S1P. SphK1 inhibition in human tonsil B cells, as well as inhibition or deletion of SphK1 in mouse splenic B cells, significantly reduced CD40-mediated Ig class switching and plasma cell differentiation ex vivo. Optimal activation of downstream CD40 signaling pathways, including NF-κB, p38, and JNK, also required SphK1. In mice treated with a SphK1 inhibitor or in SphK1(-/-) mice, isotype switching to antigen-specific IgE was decreased in vivo by 70 and 55%, respectively. Our results indicate that SphK1 is important for CD40-mediated B cell activation and regulation of humoral responses and suggest that targeting SphK1 might be a useful therapeutic approach to control antigen-specific IgE production.

Active, phosphorylated fingolimod inhibits histone deacetylases and facilitates fear extinction memory.

FTY720 (fingolimod), an FDA-approved drug for treatment of multiple sclerosis, has beneficial effects in the CNS that are not yet well understood, independent of its effects on immune cell trafficking. We show that FTY720 enters the nucleus, where it is phosphorylated by sphingosine kinase 2 (SphK2), and that nuclear FTY720-P binds and inhibits class I histone deacetylases (HDACs), enhancing specific histone acetylations. FTY720 is also phosphorylated in mice and accumulates in the brain, including the hippocampus, inhibits HDACs and enhances histone acetylation and gene expression programs associated with memory and learning, and rescues memory deficits independently of its immunosuppressive actions. Sphk2(-/-) mice have lower levels of hippocampal sphingosine-1-phosphate, an endogenous HDAC inhibitor, and reduced histone acetylation, and display deficits in spatial memory and impaired contextual fear extinction. Thus, sphingosine-1-phosphate and SphK2 play specific roles in memory functions and FTY720 may be a useful adjuvant therapy to facilitate extinction of aversive memories.

Sphingosine-1-phosphate in chronic intestinal inflammation and cancer.

Sphingosine-1-phosphate (S1P), a pleiotropic bioactive lipid mediator, and the kinase that produces it have now emerged as key regulators of numerous cellular processes involved in inflammation and cancer. Here, we review the importance of S1P in colitis and colitis-associated cancer (CAC) and discuss our recent work demonstrating that S1P produced by upregulation of SphK1 during colitis and associated cancer is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor Stat3, and consequent upregulation of the S1P receptor, S1PR1. The effectiveness of the pro-drug FTY720 (known as fingolimod), approved for the treatment of multiple sclerosis, has become the gold standard for S1P-centric drugs, and will be used to illustrate the therapeutic value of modulating SphK1 and S1P receptor functions. We will discuss our recent results showing that FTY720/fingolimod administration interferes with the SphK1/S1P/S1PR1 axis and suppresses the NF-κB/IL-6/Stat3 malicious amplification loop and CAC. These preclinical studies suggest that FTY720/fingolimod may be useful in treating colon cancer in individuals with ulcerative colitis.

Hepatic apolipoprotein M (apoM) overexpression stimulates formation of larger apoM/sphingosine 1-phosphate-enriched plasma high density lipoprotein.

Apolipoprotein M (apoM), a lipocalin family member, preferentially associates with plasma HDL and binds plasma sphingosine 1-phosphate (S1P), a signaling molecule active in immune homeostasis and endothelial barrier function. ApoM overexpression in ABCA1-expressing HEK293 cells stimulated larger nascent HDL formation, compared with cells that did not express apoM; however, the in vivo role of apoM in HDL metabolism remains poorly understood. To test whether hepatic apoM overexpression increases plasma HDL size, we generated hepatocyte-specific apoM transgenic (APOM Tg) mice, which had an ∼3-5-fold increase in plasma apoM levels compared with wild-type mice. Although HDL cholesterol concentrations were similar to wild-type mice, APOM Tg mice had larger plasma HDLs enriched in apoM, cholesteryl ester, lecithin:cholesterol acyltransferase, and S1P. Despite the presence of larger plasma HDLs in APOM Tg mice, in vivo macrophage reverse cholesterol transport capacity was similar to that in wild-type mice. APOM Tg mice had an ∼5-fold increase in plasma S1P, which was predominantly associated with larger plasma HDLs. Primary hepatocytes from APOM Tg mice generated larger nascent HDLs and displayed increased sphingolipid synthesis and S1P secretion. Inhibition of ceramide synthases in hepatocytes increased cellular S1P levels but not S1P secretion, suggesting that apoM is rate-limiting in the export of hepatocyte S1P. Our data indicate that hepatocyte-specific apoM overexpression generates larger nascent HDLs and larger plasma HDLs, which preferentially bind apoM and S1P, and stimulates S1P biosynthesis for secretion. The unique apoM/S1P-enriched plasma HDL may serve to deliver S1P to extrahepatic tissues for atheroprotection and may have other as yet unidentified functions.

Sphingosine-1-phosphate links persistent STAT3 activation, chronic intestinal inflammation, and development of colitis-associated cancer.

Inflammatory bowel disease is an important risk factor for colorectal cancer. We show that sphingosine-1-phosphate (S1P) produced by upregulation of sphingosine kinase 1 (SphK1) links chronic intestinal inflammation to colitis-associated cancer (CAC) and both are exacerbated by deletion of Sphk2. S1P is essential for production of the multifunctional NF-κB-regulated cytokine IL-6, persistent activation of the transcription factor STAT3, and consequent upregulation of the S1P receptor, S1PR1. The prodrug FTY720 decreased SphK1 and S1PR1 expression and eliminated the NF-κB/IL-6/STAT3 amplification cascade and development of CAC, even in Sphk2(-/-) mice, and may be useful in treating colon cancer in individuals with ulcerative colitis. Thus, the SphK1/S1P/S1PR1 axis is at the nexus between NF-κB and STAT3 and connects chronic inflammation and CAC.

The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 modulates cytokine expression in macrophages via p50 nuclear factor κB inhibition, in a PI3K-independent mechanism.

The Phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002 (LY2), has been previously reported to inhibit nuclear factor κB (NFκB) activity, in a PI3K-independent mechanism. The goals of the current research were to determine the specificity of LY2 regarding NFκB subunits, and to identify relevant modulation of cytokine expression in LPS-stimulated macrophages. We found that LY2 specifically diminished the level of p50, but not p65, NFκB in the nucleus of LPS-stimulated mouse RAW264.7 macrophages and human THP-1 monocytes. This activity of LY2 was mimicked by its PI3K-inert analog LY303511 (LY3), but not by another PI3K inhibitor - wortmannin. We further show that LY2 inhibited LPS-induced IL-10 expression by RAW264.7 macrophages, in a PI3K-independent mechanism. Moreover, using a deletion mutant of an IL-10 promoter reporter gene we demonstrate that the activity of the NFκB enhancer site at the IL-10 promoter is regulated by LY2 in a PI3K-independent manner. Finally, both LY2 and LY3 elevated TNFα production in the LPS tolerant state which is regulated by p50 NFκB homodimers, but not before tolerance development. The effects of LY2 and LY3 on p50 translocation and on cytokine production in LPS-stimulated macrophages are thus consistent with specific PI3K-independent inhibition of p50 NFκB homodimer activity by LY2.

Role of CREB in modulation of TNFalpha and IL-10 expression in LPS-stimulated RAW264.7 macrophages.

The role of CREB in LPS signaling is controversial. The objective of this study was to evaluate the effect of LPS on phosphorylation and transcriptional activation of CREB, in comparison to isoproterenol, a beta-adrenergic receptor agonist. We show here that LPS elevates intra-cellular cAMP level in RAW264.7 macrophages, with slower kinetics and lower magnitude than isoproterenol. The two agents stimulated CREB phosphorylation on Ser-133 to a similar extent, but with a different mechanism; rapid and mostly PKA-mediated for isoproterenol; slow and MSK1-mediated for LPS. Interestingly, LPS-stimulated phosphorylation of CREB did not result in transcriptional activation of a CRE-regulated luciferase reporter, in contrast to stimulation by isoproterenol. Furthermore, inhibitors of p38 and MSK1, but not PKA, completely blocked the production of IL-10 and TNFalpha in LPS-stimulated macrophages. Distinctively, the PKA inhibitor H89 blocked the suppressive effect of isoproterenol on TNFalpha production, as well as its stimulatory effect on IL-10 induction, in LPS-stimulated macrophages. Likewise, while over-expression of dominant negative CREB had no effect on LPS-stimulated TNFalpha production, it blocked the suppressive effect of isoproterenol on TNFalpha production in the LPS-stimulated macrophages. Our results thus indicate that PKA-mediated phosphorylation of CREB promotes TNFalpha suppression and IL-10 induction, whereas the same phosphorylation event initiated by LPS and mediated by MSK1 is non-functional for transcriptional modulation.

The ceramide-1-phosphate analogue PCERA-1 modulates tumour necrosis factor-alpha and interleukin-10 production in macrophages via the cAMP-PKA-CREB pathway in a GTP-dependent manner.

The synthetic phospho-ceramide analogue-1 (PCERA-1) down-regulates production of the pro-inflammatory cytokine tumour necrosis factor-alpha (TNF-alpha) and up-regulates production of the anti-inflammatory cytokine interleukin-10 (IL-10) in lipopolysaccharide (LPS) -stimulated macrophages. We have previously reported that PCERA-1 increases cyclic adenosine monophosphate (cAMP) levels. The objective of this study was to delineate the signalling pathway leading from PCERA-1 via cAMP to modulation of TNF-alpha and IL-10 production. We show here that PCERA-1 elevates intra-cellular cAMP level in a guanosine triphosphate-dependent manner in RAW264.7 macrophages. The cell-permeable dibutyryl cAMP was able to mimic the effects of PCERA-1 on cytokine production, whereas 8-chloro-phenylthio-methyladenosine-cAMP, which specifically activates the exchange protein directly activated by cAMP (EPAC) but not protein kinase A (PKA), failed to mimic PCERA-1 activities. Consistently, the PKA inhibitor H89 efficiently blocked PCERA-1-driven cytokine modulation as well as PCERA-1-stimulated phosphorylation of cAMP response element binding protein (CREB) on Ser-133. Finally, PCERA-1 activated cAMP-responsive transcription of a luciferase reporter, in synergism with the phosphodiesterase (PDE)-4 inhibitor rolipram. Our results suggest that PCERA-1 activates a G(s) protein-coupled receptor, leading to elevation of cAMP, which acts via the PKA-CREB pathway to promote TNF-alpha suppression and IL-10 induction in LPS-stimulated macrophages. Identification of the PCERA-1 receptor is expected to set up a new target for development of novel anti-inflammatory drugs.