British journal of pharmacology 2015 volume 172 part 7

BJP British Journal of Pharmacology DOI:10.1111/bph.13089 www.brjpharmacol.org Themed Section: Annexins VII Programme Correspondence EDITORIAL ‘Annexins’ themed section R J Flower and M Perretti R J Flower, Centre for Biochemical Pharmacology, The William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, Charterhouse Square, London, EC1M 6BQ, UK E-mail: r.j.flower@qmul.ac.uk William Harvey Research Institute LINKED ARTICLES This article is part of a themed section on Annexins VII Programme To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-7 The ‘annexins’ are an evolutionarily ancient family of monomeric proteins which are widely distributed throughout eukaryotic phyla – specifically the animal, plant and fungal kingdoms – but which are largely absent from prokaryotes and yeasts A characteristic feature of the family is the presence of an ‘annexin core domain’ which generally comprises four (occasionally more) repeating subunits of approximately 70 amino acids (the ‘annexin’ repeat) These subunits usually contain characteristic ‘type 2′ calcium binding sites although in some members of the family, these have been replaced with other motifs More than 150 annexins have been identified over 50 species Twelve proteins have been identified in humans; these are conventionally referred to as annexin (Anx) A1-13 (the Anx-A12 gene is unassigned) The descriptor ‘A’ denotes their vertebrate origin as opposed to insect, fungal, plant or protist annexins, which are denoted by ‘B’, ‘C’, ‘D’ or ‘E’ respectively Most human annexins are thought to be derived from a single ancestral gene (Anx-A13) In addition to the characteristic core domain, individual vertebrate annexins have a unique N-terminal domain of variable length This harbours motifs that can recognize and bind to other intracellular protein partners, such as those of the S100 family, and often contains residues that can be modified by post-translational processing, including phosphorylation The N-terminus is a rapidly-evolving component of these molecules and is probably responsible for the diversity of functions found within the family Structurally, in the right intracellular milieu, free annexins fold into a concave α-helical disk Calcium binding sites on the convex side facilitate the attachment of this conformer to plasma membranes or other phospholipid containing structures The N-terminal domain lies buried in the concave surface, but may ‘flip’ out in the presence of calcium making it available for binding to other partners Why are the annexins of interest to pharmacologists? © 2015 The British Pharmacological Society Whilst we are only now beginning to understand their biology, it is clear that these proteins are involved in a great number of intracellular processes such as membrane trafficking and organization as well as, surprisingly, functioning as extracellular local hormones as well The observation (for example) that extracellular Anx-A1 has striking antiinflammatory properties, and that the pharmacophore resides within a sequence in the N-terminal domain, was certainly not an intuitive finding It seems, once again, that having developed a useful motif, evolutionary pressure has adapted it again and again to fulfill other jobs There is a growing list of pathologies – ‘annexinopathies’ – associated with defects in annexin structure or function A further discovery of interest to the pharmacological community was the demonstration that several drugs related to the benzodiazepine or phenothiazine structure can bind to the core domain of these molecules modifying their behavior Whether this is relevant to the mechanism of action of any of these drugs remains to be seen Progress in the annexin field is reviewed by periodic meetings of the annexin community The 7th International Conference on Annexins was the most recent in this series It was held on the Charterhouse Square Campus of St Barts and The London School of Medicine, Queen Mary University of London in September 2013 The event was organised and hosted by the William Harvey Research Institute and supported in part by a grant from the British Pharmacological Society Some of the papers presented at this conference are collected together here in this ‘virtual’ themed issue and speak to the many roles for annexins In the first paper in this issue, Jones and his and colleagues highlight the role of annexins as prominent membrane proteins in the trematode integument and discuss the idea that these could be used to develop an immunotherapy for Schistsomiasis (Leow et al 2015) Turning to mammalian systems, Rentero’s group investigate the role of Anx-A6 – an British Journal of Pharmacology (2015) 172 1651–1652 1651 BJP R J Flower and M Perretti unusual annexin with as opposed to the more usual ‘annexin repeats’ – as a membrane scaffolding protein and the implications of this for regulation of signal transduction (Alvarez-Guaita et al 2015) Anx-A2 is one of those annexins with confirmed extracellular as well as intracellular actions (it can act as a cell surface receptor for tissue plasminogen activator) and in the final paper, Dekker’s laboratory has used a ‘toolbox’ of peptides to study the interaction of Anx-A2 with its principal binding partner (S100A10) and to determine how this modifies and regulates its properties (Liu et al 2015) We hope that the papers published in this themed section will serve to stimulate interest in this protein family, which we commend as a fertile area for future pharmacological investigation References Liu Y, Myrvang HK, Dekker LV (2015) Annexin A2 complexes with S100 proteins: structure, function and pharmacological manipulation Br J Pharmacol 172: 1664–1676 Further reading Blackwood RA, Hessler RJ (1995) Effect of calcium on phenothiazine inhibition of neutrophil degranulation Journal of leukocyte biology 58 (1): 114–118 D’Acquisto F, Perretti M, Flower RJ (2008) Annexin-A1: a pivotal regulator of the innate and adaptive immune systems Br J Pharmacol 155 (2): 152–169 Gerke V, Creutz CE, Moss SE (2005) Annexins: linking Ca2+ signalling to membrane dynamics Nature reviews Molecular cell biology (6): 449–461 Gerke V, Moss SE (1997) Annexins and membrane dynamics Biochimica et biophysica acta 1357 (2): 129–154 Alvarez-Guaita A, Vilà de Muga S, Owen DM, Williamson D, Magenau A, García-Melero A et al (2015) Evidence for annexin A6-dependent plasma membrane remodelling of lipid domains Br J Pharmacol 172: 1677–1690 Iglesias JM, Morgan RO, Jenkins NA, Copeland NG, Gilbert DJ, Fernandez MP (2002) Comparative genetics and evolution of annexin A13 as the founder gene of vertebrate annexins Molecular biology and evolution 19 (5): 608–618 Leow CY, Willis C, Hofmann A, Jones MK (2015) Structure–function analysis of apical membrane-associated molecules of the tegument of schistosome parasites of humans: prospects for identification of novel targets for parasite control Br J Pharmacol 172: 1653–1663 Perretti M, D’Acquisto F (2009) Annexin A1 and glucocorticoids as effectors of the resolution of inflammation Nature reviews Immunology (1): 62–70 1652 British Journal of Pharmacology (2015) 172 1651–1652 BJP British Journal of Pharmacology DOI:10.1111/bph.12898 www.brjpharmacol.org Themed Section: Annexins VII Programme Correspondence REVIEW Structure–function analysis of apical membrane-associated molecules of the tegument of schistosome parasites of humans: prospects for identification of novel targets for parasite control Malcolm K Jones, School of Veterinary Sciences, The University of Queensland, Gatton, Qld 4343, Australia E-mail: m.jones@uq.edu.au; or Andreas Hofmann, Structural Chemistry Program, Eskitis Institute, Griffith University, N75 Don Young Road, Nathan, Qld 4111, Australia E-mail: a.hofmann@griffith.edu.au Received 11 December 2013 Revised 12 June 2014 Accepted 26 August 2014 Chiuan Yee Leow1,2,3, Charlene Willis2,4, Andreas Hofmann4,5 and Malcolm K Jones1 School of Veterinary Science, The University of Queensland, Gatton, Queensland, Australia, Infectious Diseases, QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia, Institute for Research in Molecular Medicine, Universiti Sains Malaysia, Penang, Malaysia, Structural Chemistry Program, Eskitis Institute, Griffith University, Brisbane, Queensland, Australia, and 5Faculty of Veterinary Science, The University of Melbourne, Parkville, Victoria, Australia Neglected tropical diseases are a group of some 17 diseases that afflict poor and predominantly rural people in developing nations One significant disease that contributes to substantial morbidity in endemic areas is schistosomiasis, caused by infection with one of five species of blood fluke belonging to the trematode genus Schistosoma Although there is one drug available for treatment of affected individuals in clinics, or for mass administration in endemic regions, there is a need for new therapies A prominent target organ of schistosomes, either for drug or vaccine development, is the peculiar epithelial syncytium that forms the body wall (tegument) of this parasite This dynamic layer is maintained and organized by concerted activity of a range of proteins, among which are the abundant tegumentary annexins In this review, we will outline advances in structure–function analyses of these annexins, as a means to understanding tegument cell biology in host–parasite interaction and their potential exploitation as targets for anti-schistosomiasis therapies LINKED ARTICLES This article is part of a themed section on Annexins VII Programme To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-7 Abbreviations Anx, annexin; NTDs, neglected tropical diseases; RA, radiation attenuated; Sm, Schistosoma mansoni; TEMs, tetraspanin-enriched microdomains; TSP, tetraspanin © 2014 The British Pharmacological Society British Journal of Pharmacology (2015) 172 1653–1663 1653 BJP C Y Leow et al Table of Links TARGETS Other protein targetsa Transportersb Enzymesc Heat shock proteins Calcium pump Calpain This Table lists key protein targets in this document, which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,b,cAlexander et al., 2013a,b,c) Neglected tropical diseases (NTDs) NTDs include some 17 lesser known chronic infections that affect poor and disenfranchised people, primarily, but not exclusively, in developing nations (Hotez et al., 2007; Hotez and Fenwick, 2009) Chronic infections caused by NTDs lead to many adverse outcomes in affected populations and contribute substantially to human morbidity In addition to microbial and protozoan diseases, NTDs include a number of helminth infections, such as diseases caused by flatworm parasites, notably schistosomiasis, echinococcosis and liver fluke diseases, as well as roundworm parasites, such as the major soil transmitted helminth infections (ascariasis, trichuriasis and hookworm diseases) Although no individual NTD rivals the major infectious threats of HIV, malaria or tuberculosis in terms of global impact of disease, collectively, the NTDs contribute substantially to morbidity throughout the world (Engels and Savioli, 2006) A number of factors present major challenges for the development of new treatments for NTDs Firstly, NTDs are chronic diseases, which may reside in affected people as lifelong infections Secondly, NTDs are not always associated with human mortality and the burden of these diseases can be subtle, hidden among such other measures of disease burden as hindered development, poor cognitive function and chronic ailments Thirdly, as stated, NTDs often affect the poorest of the poor, people often unable to pay for medical treatments, especially for chronic illnesses Hence, these diseases receive less attention than other, immediately lifethreatening infectious diseases Lastly, poor development of infrastructure systems in impoverished countries also irreversibly impacts on efficient drug distribution for the treatment of these NTDs (Chimbari et al., 2004) Among the NTDs of major interest is the suite of diseases known as human schistosomiasis These diseases are caused by infection with any of a number of species of the genus Schistosoma, a taxon of platyhelminth trematodes, commonly known as blood flukes (and historically, known as the agents of bilharzia) (Ross et al., 2002) Five species are the main contributors to human schistosomiasis, Schistosoma mansoni (Sm), S japonicum, S mekongi, S intercalatum and S haematobium Transmission of the parasites to humans takes place in freshwater, typically in regions of poor sanitation where human excreta contaminate water bodies The egg hatches in freshwater to liberate a larva, which searches for and infects a species of snail Schistosomes, like other trema1654 British Journal of Pharmacology (2015) 172 1653–1663 todes, display high host specificity for their snail host The distribution of a schistosome species is largely dependent on the geographic distribution of its snail host Schistosomes infect over 200 million people in approximately 74 nations, the majority of which are in Sub-Saharan Africa and the Middle East (Steinmann et al., 2006) Distinct foci of schistosomiasis also occur in Asia (China, the Philippines, along the Mekong River and Indonesia), as well as South America (notably Brazil and some Caribbean Islands) Disability-adjusted life years lost to human schistosomiasis in 2010 were measured at 48/100 000, an increase of 20% on estimates made in 1990 (Murray et al., 2013) There is a distinct dichotomy in schistosomiasis in relation to the host responsiveness to various life stages On the one hand, the invasive larvae and adult parasites are largely able to avoid immunosurveillance of the hosts To that effect, these parasites employ a series of strategies including rapid development, stealth-like host interfaces and immunosuppression (Wilson, 2009) On the other hand, the active secretion of immunogenic molecules by eggs provokes an intense immune response (Burke et al., 2009) This phenomenon is characterized by a strong granulocytic response around the egg in affected tissues that may lead to fibrosis, particularly in the liver The intense response enables the escape of the eggs from the host The cellular infiltrate forces a schistosome egg across the vascular endothelium and into tissues of luminal organs, such as the intestinal lining, the bladder wall or genital organs, driving the egg ultimately into the lumen, from which the egg is voided into the environment The bulk of chronic disease in schistosomiasis is related to host responses against parasite eggs deposited in the blood vessels surrounding the gut (Sm and S japonicum) or bladder and genital organs (S haematobium) (Ross et al., 2002) However, it has proven more effective to direct control towards killing adult worms or the invasive larvae that establish infection so that the deposition of eggs is stopped Schistosomes belong to the Clade Lophotrochozoa of the Kingdom Animalia The multicellular animals are monophyletic (Walker et al., 2011) and there are substantial similarities among the many cellular, biochemical and molecular adaptations in different animal clades The search for effective treatments against schistosomiasis thus needs to exploit key molecular and conformational differences between target molecules of these parasites and their hosts This review explores work focused on the search for novel molecular targets of therapeutics and prophylactics, and examines new Annexins of schistosomes insights from studies of a primary site of host interaction, the schistosome tegument Some annexins of schistosomes are abundant molecules in the proteome of the schistosomes These proteins are found in close association with the apical membrane of the tegument of the parasites In view of their abundance, distribution and distinctive structure, these proteins are of interest, both as targets and as vehicles to understand the dynamic nature of the apical membrane complex of these parasites, a complex that is crucial for survival of the parasites in their hosts Treatments for schistosomiasis – drugs There currently exist few drugs for treatment of schistosomiasis: praziquantel, metrifonate, oxamniquine and artemether (Cioli et al., 1995; Ross et al., 2002; Bartley et al., 2008) All of these drugs have proven useful for therapeutic treatment of individuals in the clinic or of communities in mass drug administration Of the four drugs, oxamniquine is only effective against schistosomiasis mansoni; resistance to this drug by the parasite is known and the mechanism of resistance elucidated (Valentim et al., 2013) Metrifonate is only effective against urinary schistosomiasis, caused by S haematobium, and its use is hampered by a complex administration schedule with multiple doses required over a week period Frequently, this therapy is met with a low rate of compliance among patients Furthermore, the drug is labile in warm climates and is thus less useful in field settings Combination therapy using praziquantel and metrifonate has been effective for urinary schistosomiasis (Danso-Appiah et al., 2009) Artemether is a β-methyl ether derivative of artemisinin, a compound derived from the sweet wormwood Artemesia annua Artemisinin and its derivatives are highly effective against haematophagous parasites, notably malaria, but they have also proven effective against schistosome infection (Liu et al., 2012) One recent report suggests that artemisinin is acted upon by elemental iron in the iron-rich environment of haematophagous parasites and the complex, in turn, inhibits calcium transport (Shandilya et al., 2013) Concerns about resistance to artemisinin and its derivatives by the more insidious human disease of falciparum malaria has precluded the use of artemether against schistosomiasis where the two diseases are co-endemic (Bergquist et al., 2005; Utzinger et al., 2007) The current drug of choice for treatment of schistosomiasis is praziquantel This drug has been used in mass treatment campaigns in many countries and remains a primary tool in the war against the disease (Knopp et al., 2013) The mode of action of praziquantel remains unknown, although recent developments strongly suggest a role for the drug in calcium homeostasis in the parasites and notably in calcium transport complexes (Greenberg, 2005; You et al., 2013) The drug remains highly effective for a wide range of flatworm diseases of humans and domestic animals Praziquantel has been deployed for mass drug administration in endemic regions and has been successful in pushing the disease from high to low endemicity (Geary, 2012) This major achievement has been facilitated in part by reductions in costs associated with manufacture of the drug, and the development of public– private partnerships that have led to the distribution of the BJP drug to many impoverished communities where schistosomiasis is endemic Despite its high efficacy, praziquantel has limitations (Geary, 2012) The drug is only effective against adult or pre-adult forms (Greenberg, 2005) Furthermore, praziquantel confers no protection against subsequent infection and people may become reinfected within days of treatment (Ross et al., 2002) Treatment failures for S mansoni and S haematobium infections have been observed, and the presence of resistant strains has been demonstrated experimentally (Greenberg, 2013) Although widespread resistance to praziquantel has not been observed clinically, the application of the drug in mass treatment campaigns may result in new resistant forms emerging and new replacement drugs and formulations are needed (Geary, 2012) Prevention of schistosomiasis – vaccines Many experts within the schistosomiasis community argue that continued application of a single drug, praziquantel, for single treatments and as a mass control strategy is problematic and not likely to lead to effective control of the disease The alternative, a subunit vaccine, has thus been promoted as an important alternative strategy for the control and elimination of schistosomiasis (Bergquist et al., 2008; McManus and Loukas, 2008; Loukas et al., 2011; Kupferschmidt, 2013) Optimism for a vaccine rests on observations from the 1970s on host responses to radiation-attenuated (RA) cercariae in experimental infections (Bickle et al., 1979a,b) A cercaria is the larval stage that penetrates human skin to initiate infection This stage transforms rapidly in human skin to become a host-adapted larva, the schistosomulum This larva then follows a set pattern of migration and development over the following days and weeks, passing along vasculature through the lung and liver In the liver, a male parasite will mate with a female and carry her to mesenteric or pelvic circulation, the final destination being parasite species specific (Wilson, 2009) It was shown that infection of humans with live, RA parasites led to strong protection against subsequent challenge infections with normal cercariae (Correa-Oliveira et al., 2000; Ribeiro de Jesus et al., 2000) Vaccination of animal models with RA cercariae has thus led to an adult worm burden reduction in experimental schistosomiasis of 60–70% (Bickle et al., 1979a,b; CauladaBenedetti et al., 1991; Coulson et al., 1998; McManus, 1999; Dillon et al., 2008) The molecular mechanism of protection with RA is unclear; however, the immune response appears to result from transcriptional suppression in the attenuated parasites during the early stage of development (Dillon et al., 2008) Transcriptional suppression in RA was observed for a variety of genes including those encoding tegument proteins, members of signalling pathways associated with GPCRs, neurotransmitters and cytoskeletal components The major lessons learned from these studies are that parasite killing is largely dependent on host–parasite interaction during the host establishment phase of the parasites, that is, within the first week after infection During this time, the cercaria undergoes an extensive remodelling of its surface body wall, the British Journal of Pharmacology (2015) 172 1653–1663 1655 BJP C Y Leow et al tegument and becomes transcriptionally active for a series of molecules associated with surface dynamics and nutrient absorption (Gobert et al., 2009b), compared with the cercaria Indeed, some of the promising vaccine candidates come from this tissue, and it seems that vaccine targeting of this layer is crucial for parasite killing Despite the high level of protection available with radiation-attenuated vaccines, the unstable lifespan, delivery problems and safety problems of these modified cercariae makes them unsuitable for further development as a vaccine (Bergquist et al., 2008) Therefore, efforts have been directed to discover and identify suitable protective antigens from schistosomes, leading to the development of recombinant vaccines, DNA vaccines, peptide–epitope-based vaccines, multivalent vaccines and chimeric vaccines (McManus and Loukas, 2008) Of the vaccines trialled, a number have been promoted for human trials, including the Bilvax vaccine based on a 28 kDa S haematobium glutathione-S-transferase, which has entered phase clinical trials, and a S mansoni tetraspanin (Sm-TSP-2) (Tran et al., 2006), which has entered phase trials (Loukas et al., 2011; Kupferschmidt, 2013) Other vaccines presented at a recent vaccine discovery workshop sponsored by the Bill and Melinda Gates Foundation in the United States (Kupferschmidt, 2013) identified additional vaccines still in experimental development, including Sm14, a fatty acid binding protein, a calpain (Smp80) from S mansoni, and Sj23, a TSP, a triose-phosphate isomerase, an insulin receptor, and paramyosin from S japonicum (Zhu et al., 2004; 2006; Siddiqui et al., 2005; Tendler and Simpson, 2008; You et al., 2012) An advantage of vaccination strategies against the zoonotic S japonicum is that the parasite is found in a variety of domesticated animals, including water buffalo and goats in China Researchers involved in controlling this species in China and the Philippines have developed vaccines for use in animals as transmission-blocking vaccines, based on modelling of transmission dynamics in endemic regions (McManus et al., 2009) Antigen discovery studies are still progressing using a variety of immunomics and proteomic approaches It is now widely appreciated that targeted approaches are required for antigen discovery, and there is continuing interest in considering fundamental cell biological and developmental understanding with molecular advances The tegument of schistosomes The tegument, or body wall, of schistosomes is a dynamic host-adapted interface between the parasite and its vascular environment The tegument is a highly polarized syncytium and possesses functional analogy with transporting epithelia, including the gut lining or the syncytiotrophoblasts of the human placenta The tegument plays significant roles in nutrient uptake, immune evasion and modulation, excretion, osmoregulation, sensory reception, and signal transduction (Jones et al., 2004; Kusel et al., 2007; Castro-Borges et al., 2011) Given the importance of the schistosome tegument in nutrition and immune evasion, proteins of this surface layer are recognized as prime candidates to target for vaccine and therapeutic drug development (Loukas et al., 2007) 1656 British Journal of Pharmacology (2015) 172 1653–1663 Ultrastructure of schistosome tegument The tegument is formed as a single syncytium that covers the entire body and is continuous with other epithelia (Figures 1– 2), notably the foregut lining (Silk et al., 1969) This surface cytoplasmic layer is a highly ordered structure with distinct transporting regions, secretory components and absorptive adaptations A peculiarity of the layer is the presence of a dual membrane complex that forms the apical extremity of the tegument cytoplasm (Hockley, 1973; Hockley and McLaren, 1973; Castro-Borges et al., 2011) The developmental activity of cercarial transformation referred to above appears first and foremost to involve alteration of the apical membrane of these parasites soon after invasion (Hockley and McLaren, 1973; Skelly and Shoemaker, 1996; 2001; Keating et al., 2006) The single-unit membrane of the cercaria, with its highly immunogenic glycocalyx, becomes replaced by a host-adapted dual membrane system, consisting of the membrane proper, overlain by an additional unit membrane, the membranocalyx Although the membranocalyx is depauperate of parasite-derived proteins, the underlying membrane is decorated with abundant membrane proteins (Braschi and Wilson, 2006) Membrane repair and maintenance is an ongoing process, as evidenced by abundant cytoplasmic inclusions and molecule associated with the apical membranes The advantage to schistosomes in possessing a syncytial tegument is poorly understood, but appears to be an important strategy that ensures survival of parasites in the vascular environment Invaginations of the surface membrane complex, as well as in the basal membrane of the cytoplasm (Hockley, 1973; Gobert et al., 2003; Skelly and Wilson, 2006), are structural evidence of high turnover of these membranes (Brouwers et al., 1999), a process that is related to nutrient uptake and a way of avoiding the host immune response by internalizing antibodies and removing possible antigenic molecules from the surface (Skelly and Wilson, 2006) Membrane internalization and translocation events are driven by a complex interplay of multiple membrane proteins including the TSP-enriched microdomains (TEMs) (Tran et al., 2010; Jia et al., 2014) The TEMs are protein complexes formed about a membrane-resident TSPs, which act as scaffold proteins for the multiple fusion and scission activities of plasma membrane (Hemler, 2008) For S.mansoni, TEM residents include a variety of proteins strongly linked to the apical plasma membrane, including schistosome annexins B30, Sm29, a dysferlin, calpain, fructose-biphosphate aldolase, heat shock protein 70 and actin (Jia et al., 2014) The tegument is supported by cell bodies that lie embedded in the parasite parenchyma (Hockley, 1973; Gobert et al., 2003) The apical cytoplasm of the tegument and the cell bodies are linked by cytoplasmic bridges, which traverse the muscle bundles lying beneath the parasite tegument (Figure 1B) (Hockley, 1973; Gobert et al., 2003) Tegumentary cell bodies contain the synthetic machinery of the syncytium, including endoplasmic reticulum and Golgi apparatus, and produce abundant vesicular products that are trafficked to the tegument along cytoplasmic bridges (Figure 3) The molecular interactions driving membrane formation during transformation and in repair and renewal are far from understood, but the abundance of adaptor and chaperone Annexins of schistosomes BJP Figure Tegument of Schistosoma mansoni by transmission electron microscopy (A) Low magnification image from a cross-section of an adult The image shows the apical cytoplasm of the tegument (Teg), which is the interface with the host vasculature The syncytial cytoplasm rests on bands of musculature and is supported and maintained by tegumentary cell bodies That depicted is rich in vesicles that are transported to the apical cytoplasm along cytoplasmic bridges (arrows) The parasite digestive system, lined by a syncytial epidermis called a gastrodermis, lies deep within the body (B) High magnification view of the teguments of paired male and female adults The apical membrane complex (AP) consists of a plasma membrane overlain by a subsidiary membrane-like structure, the membranocalyx, evident only after special fixation/staining of TEM tissues using uranyl acetate The apical cytoplasm infolds frequently as surface invaginations, sometimes with secondary caveola-like outpocketings appearing Other bodies decorate the tegument, including discoid bodies (DB) Tegumentary spines (SP), used for adhesion, are observed Original figures proteins associated with the apical membrane complex (Table 1) and abundance of membrane vesicles (Figures 1–2) suggest a continuous cycle of renewal and repair throughout adult life of the parasite Tegument proteins as vaccine targets Figure Immuno-electron microscopy of Sm-Anx-B22, transmission electron microscopy, using indirect immunocytochemistry incorporating 10 nm protein-A gold particles Sm-Anx-B22 was localized to surface invaginations (SI) and other membrane compartments associated with the apical plasma membrane complex (AP) After Leow et al (2014) In mice immunized with tegument extract of newly transformed S mansoni schistosomula, an induced Th1-type protection has been observed, which damages the adult worm tegument layer and reduces egg number and parasite burden in challenge infections (Smithers et al., 1990) Therefore, it is currently believed that tegument proteins of schistosomes are a priority in antigen discovery Proteins potentially exposed at its surface during intra-mammalian stages are possibly the most susceptible targets for vaccine development (Loukas et al., 2007) A challenge in studies of schistosome biology is the elucidation of when and how during the infection targets are exposed to the host immune recognition According to models of the S mansoni tegument, the primary vaccine target Sm-TSP-2 (tetraspanin-2) occurs in the plasma membrane, that is, it lies hidden from the host under the membranocalyx (Wilson, 2012) Recent immunolocalization data suggest the molecule is even more hidden from the host, in adult parasites at least, occurring predominantly in association with surface invaginations of the hosts and in subsidiary membranes (Schulte et al., 2013) British Journal of Pharmacology (2015) 172 1653–1663 1657 BJP C Y Leow et al Figure Cartoon representations of (A) human annexin A5, (B) α1-giardin from Giardia intestinalis and (C) the dimerized Schistosoma mansoni annexin Sm-Anx-B22 For annexin A5 and α1-giardin, the annexin repeats are shown in different colours For all annexins shown the N-terminus is coloured blue and the II/III linker in magenta Note the distinctly longer linker in Sm-Anx-B22, which packs against the N-terminal domain The disulphide bond between Cys173 (molecule 1) and Cys173 (molecule 2) is rendered in green Protein structures were rendered with PyMOL (DeLano, 2002) Analysis of the schistosome proteome has vastly increased the speed of identification of tegument proteins (Braschi and Wilson, 2006; Mulvenna et al., 2010; Castro-Borges et al., 2011; Jia et al., 2014) (Table 1) A series of experiments have allowed different proteins to be assigned to the distinct membrane fractions of the apical membrane complex (Wilson, 2012), although these assignments are likely to be crude and require further analysis by refined localization tools A range of molecules has been identified including glucose transporters, proteases and other enzymes, receptors, chaperones and structural proteins (Mulvenna et al., 2010; Castro-Borges et al., 2011; Wilson, 2012) Further confirmation of the co-location of many of these molecules has come from interaction studies of Sm-TSP-2 (Jia et al., 2014), which show strong interactions, as stated, with a range of surface-linked molecules including annexin B30, alkaline phosphatase, actin, an aldolase, calpain, HSP70, dysferlin and Sm29, a schistosome-specific molecule These interacting partners widen the pool of available molecules for vaccination studies Among the dominant surface-related proteins is S mansoni annexin B30 (hereafter Sm-Anx-B30) (CastroBorges et al., 2011; Cantacessi et al., 2013; Jia et al., 2014) This molecule is strongly associated with the tegument (Tararam et al., 2010) and the Sm-TSP-2 TEMs, although how it binds to other proteins is undetermined Sm-Anx-B30 lies in direct association with the apical plasma membrane (C Leow, unpubl obs.) Three other S mansoni annexins, namely, Sm-Anx B7a, B22 and B5a, have been shown in various studies of different schistosomes to be located to the tegument The abundance, as well as the peculiar features of annexins of some parasite groups, makes them potential targets for therapies Schistosome annexins Annexins are a family of proteins that are able to bind to acidic phospholipid membranes Their membrane-binding mode includes formation of a ternary complex involving the protein, the calcium ions and the membrane The survey of group B annexins from different invertebrate taxa revealed that the proteins occur in the vast majority of species studied so far (Cantacessi et al., 2013) The abundant annexin proteins are conspicuously evident in many parasite groups, 1658 British Journal of Pharmacology (2015) 172 1653–1663 including a series of arthropod vectors of disease, as well as basal metazoans, but are apparently absent from others, notably the Mollusca Using structure-based amino acid sequence alignments and phylogenetic analyses, the recent analysis provided a robust classification for this protein group, enabling information on structure–functional relationships of these proteins, as well as to assign names to sequences with ambiguous annotations in public databases (Cantacessi et al., 2013) It was immediately apparent in phylogenetic analyses that gene duplication in divergent clades was the major evolutionary event in annexins’ genesis, particularly in schistosomes The highest representation of annexin was found in S mansoni with 13 annexins, many distributed on two chromosomes, suggesting linkage (Cantacessi et al., 2013) Evidence gained from tissue-specific transcriptional and proteomic profiling of adult parasites suggests that the different schistosome annexins are expressed differentially throughout the body of the parasites (Gobert et al., 2009a) As stated, Sm-Anx-B7a, B22 and B30 are distinctly associated with the syncytial tegument (Braschi and Wilson, 2006; Mulvenna et al., 2010) Our tissue-specific transcriptomic survey of female S japonicum indicated that different annexins were expressed preferentially by different cell types, with the gut lining expressing annexin B7 and B22, while the vitelline gland expressed annexin B5 (Gobert et al., 2009a) Although S japonicum is a distinctive parasite, as it diverged early from other species of Schistosoma, similar patterns of annexin expression might reasonably be expected to be conserved within the genus The abundance of annexin B7 and B22 in gut and tegument allows the postulate that these molecules may be epithelial annexin in these parasites, and thus associated with syncytial epithelia Importantly, both the gastrodermis and the tegument are predicted to have high membrane turnover and reshaping (Nawaratna et al., 2011) Structure–function observations of schistosome annexins Observations made by us and others in the recent past point towards a potential use of parasite annexins as therapeutic Annexins of schistosomes BJP Table Apical membrane complex-associated proteins of schistosomes Membranocalyx associated Inter-membrane space Plasma membrane Proteins Putative role Tetraspanin-2 Mediators of tetraspanin-enriched microdomain in surface membrane complex; membrane remodelling kDa low-molecular weight protein? Secreted protein Sm13 Uncertain, membrane protein Sm29 Uncertain role, but potential ligand of tetraspanin Sm200 Uncertain role Bound host proteins-CD48, 90, immunoglobulins, complement factors Host molecules kDa low-molecular weight protein? Secreted protein Annexin B30 Membrane maintenance Alkaline phosphatase Hydrolase Carbonic anhydrase Metalloenzyme kDa low-molecular weight protein? Secreted protein CD59 Complement inhibition PDE PDE Apyrase Apyrase Tetraspanins Membrane recycling, tetraspanin-enriched microdomains Glucose transporter (SGTP-4) Glucose uptake Aquaporins Water and small solute transport Na/K ATPase Na/K ATPase Anion channel Cytosolic Plasmolipins Tetraspanin myelin proteins, membrane rafts Dysferlin Calcium-dependent membrane fusion events; component of tetraspanin-enriched microdomains in tegument Calpain? Protease Tegument allergen-like proteins (Sm 22.6, Sm21.7) Dynein-like motor function; calcium binding GAPDH GAPDH Heat shock protein (HSP70) Chaperone Actin Cytoskeleton Dynein light chains Dynein-like motor function Evidence or proposed location in the tegument is derived from the review of proteomic analyses of the tegument of Schistosoma mansoni by Wilson (2012) In many cases, the link between the protein and unit membrane is inferred and further experimental evidence is required The model is based on a static two-dimensional structure only and ignores the dynamic nature of the schistosome tegument The membrane complex is a dual membrane system, consisting of a unit membrane overlaid by an additional membrane structure, the so-called membranocalyx Only one annexin is listed here, although it is known that multiple annexins are present in the tegument of schistosomes targets These findings include (i) immunoreactivity of some parasite annexins (Hongli et al., 2002; Palm et al., 2003; Weiland et al., 2003; Gao et al., 2007; Weeratunga et al., 2012; Leow et al., 2014); (ii) localization of certain parasite annexins to areas of potential exposure and/or structural integrity (Braschi and Wilson, 2006; Jia et al., 2014); and (iii) the existence of a unique structural feature, including the extended helical linker between repeats II and III (Figure 3), in parasite annexins that differentiates them from host annexins (Hofmann et al., 2010; Weeratunga et al., 2012; Leow et al., 2014) The extended linker region is a primary source of variation between some group B and group A annexins Many group B annexins, including those from the cestode Taenia solium; annexin B36 (nex-4) from the model nematode Caenorhabditis elegans; and some Group E annexins, including α-12- and α-19 giardin, possess an unusually long linker segment between repeats II and III on the concave side of the protein (Hofmann et al., 2010) (Figure 3) Whereas the typical length of this linker in annexin ranges from 10 to 15 amino acids, the linker peptide of these groups B and E range from 25 to 38 amino acids (Hofmann et al., 2010) Secondary structure predictions consistently indicate that this elongated linker region adopts an α-helical structure, and the recent crystal structure of Sm-Anx-B22 provided the anticipated experimental proof (Leow et al., 2014) We hypothesize that British Journal of Pharmacology (2015) 172 1653–1663 1659 BJP C Y Leow et al Figure Surface-rendered model of Schistosoma mansoni annexin Sm-AnxB22 A distinctive groove, lying at the junction of the two partners of the dimer (coloured light blue and tan) may give the protein an adaptor function The N-terminus (dark blue) and II/II linker region (magenta) of one partner are shown Figure prepared with PyMOL (DeLano, 2002) this additional α-helical element on the concave side of the molecule may provide a target for immunological therapeutics (Hofmann et al., 2010) It is tempting to speculate that other parasite annexins with an extended II/III linker peptide may adopt a very similar conformation A comparison of the extent of the N-terminal domains for annexins with the unique linker shows that such a fold may be possible for most of them The crystal structure of Sm-Anx-B22 confirms the presence of the predicted α-helical segment in the II/III linker and also reveals a covalently linked head-to-head dimer (Leow et al., 2014) Sm-Anx-B22 and its homologues from S japonicum (Cantacessi et al., 2013) and S bovis (de la Torre-Escudero et al., 2012) are the only B annexins known to date that possess an exposed cysteine residue in the IIDE loop (Cys173), a position where most other annexins possess a serine residue In Sm-Anx-B22, the involvement of Cys173 in an inter-molecular disulphide bond as well as several intimate electrostatic side chain interactions add to the stabilization of the unique head-to-head dimer topology where the dimer interface is exclusively located in module II/III Structurally, this is significantly different to other annexin headto-head dimers (Hofmann et al., 2010), where the dimer interface comprises the entire convex surface of both molecules In addition, from the calcium-bound crystal structure of Sm-Anx-B22, canonical as well as novel calcium binding sites can been identified, which seems to be a recurring motif in parasite annexins Intriguingly, the dimer arrangement observed in the annexin B22 crystal structure revealed the presence of two non-anticipated prominent features: a potential non-canonical membrane-binding site and a potential binding groove opposite of the former (Figure 4) Annexins in schistosomes A variety of roles have been proposed for annexins In vertebrates, annexins are known to display a broad range of 1660 British Journal of Pharmacology (2015) 172 1653–1663 biological activities including response to inflammation, membrane traffic and adhesion, anticoagulation, signal transduction, developmental processes and membrane repair (Bouter et al., 2011; Draeger et al., 2011) In parasites, annexins are suggested to be involved in maintenance of membrane structure (Peattie et al., 1989; Tararam et al., 2010), anti-inflammatory activity (Zhang et al., 2007) and fibrinolytic activity (de la Torre-Escudero et al., 2012) Annexins may thus have distinct roles in enabling survival of parasites when they are within the hosts Some annexins are speculated to be involved in redox reactions and the regulation of reactive oxygen molecules in plants (Hofmann et al., 2003) (Konopka-Postupolska et al., 2011) and in mammals (Tanaka et al., 2004; Madureira et al., 2011; Madureira and Waisman, 2013) Localization of Sm-Anx-B22 by fluorescence and electron microscopy in different species of schistosomes (Tararam et al., 2010; de la Torre-Escudero et al., 2012; Leow et al., 2014) demonstrates that the molecule is strongly associated with the tegument and the plasma membrane structures of the apical regions of the tegument of adult parasites (Figure 2) The molecule is expressed in human-parasitic phases of the parasite life cycle, suggesting a major role in surface membrane dynamics during life in the human host Although Sm-Anx-B22 shares many structural similarities with other annexins, its dimeric nature as well as the unique extended linker region suggests that this molecule is co-adapted to function in the peculiar syncytial environment of the tegument of these parasites (Leow et al., 2014) Among the peculiarities, there is a prominent groove that occurs within the dimeric species (Figure 4) This groove is postulated to enable the Sm-Anx-B22 dimer to assume an adaptor function, linking the apical membrane complex with proteins Sm-Anx-B22 possesses another unique feature, namely, the external arrangement of the II/III linker that is reflexed over the N-terminal region of the molecule There is now substantial evidence that annexins, and indeed other molecules of the schistosome tegument, adopt unique conformations that might be exploited for therapeutics or prophylaxis as they distinguish the parasite proteins from homologous proteins in the host The question remains as to how these unique regions might be targeted if we are to develop anti-schistosomiasis therapies directed against these molecules Undoubtedly, as with all areas of investigation concerning annexins from a wide variety of organisms, more structure–function analyses of the proteins in cells is required For schistosomes the peculiarities of the annexins, including the extended II/III linker regions, non-canonical calcium binding sites and other molecular anomalies are of interest, not only for enhancing fundamental understanding of membrane dynamics, but also for designing anti-parasite targets Being highly adapted to life within hosts, helminth parasites present considerable difficulties in functional genomics analyses They are not easily cultivated outside of the host and require molecular signalling from their host to develop fully Furthermore, transgenesis studies for schistosomes remain in their infancy, although these parasites are amenable to RNA-interfering technologies Thus, studies of annexins of schistosomes present some challenges Two important BJP W-C Chen et al Figure WMJ-S-001 suppressed COX-2 and iNOS expression in LPS-stimulated RAW264.7 macrophages (A) Chemical structures of WMJ-S-001, WMJ-S002, WMJ-S-003 and WMJ-S-004 (B) Cells were pretreated with 10 μM of WMJ-S-001, WMJ-S-002, WMJ-S-003 or WMJ-S-004 for 30 followed by the stimulation with LPS (1 μg·mL−1) for another 24 h After treatment, cells were harvested to assess the COX-2 and iNOS levels by immunoblotting Each column represents the mean ± SEM of at least four independent experiments *P < 0.05, compared with the control group; # P < 0.05, compared with the group treated with LPS alone (C) Cells were pretreated with the vehicle or WMJ-S-001 at the indicated concentrations for 30 before treatment with LPS (1 μg·mL−1) for another 24 h The COX-2 and iNOS levels were then determined by immunoblotting Each column represents the mean ± SEM of six independent experiments *P < 0.05, compared with the control group; #P < 0.05, compared with the group treated with LPS alone (D) Cells were transfected as described in (C); media were then collected to measure PGE2 as described in the ‘Methods’ section Each column represents the mean ± SEM of three independent experiments performed in duplicate *P < 0.05, compared with the control group; #P < 0.05, compared with the group treated with LPS alone (E) Cells were treated with WMJ-S-001 at the indicated concentrations for 24 h, cell viability was determined using a trypan blue dye exclusion test Each column represents the mean ± SEM of four independent experiments 1896 British Journal of Pharmacology (2015) 172 1894–1908 WMJ-S-001 suppresses LPS-induced COX-2 expression Methods Synthesis of WMJ-S WMJ-S compounds were synthesized as described in the Supporting Information Cell culture The RAW 264.7 mouse macrophage cell line was purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan), and cells were maintained in DMEM (Life Technologies, Grand Island, NY, USA) containing 10% FBS, 100 U·mL−1 of penicillin G, and 100 μg·mL−1 streptomycin in a humidified 37°C incubator Immunoblotting Immunoblot analysis was performed as described previously (Huang et al., 2013) Briefly, cells were lysed in an extraction buffer containing 10 mM Tris (pH 7.0), 140 mM NaCl, mM phenylmethanesulfonylfluoride, mM DTT, 0.5% NP-40, 0.05 mM pepstatin A and 0.2 mM leupeptin Samples of equal amounts of protein were subjected to SDS-PAGE and transferred onto an nitrocellulose membrane, which was then incubated in a tris-buffered saline and tween 20 buffer containing 5% non-fat milk Proteins were visualized by incubating with specific primary antibodies for h followed by HRP-conjugated secondary antibodies for another h Immunoreactivity was detected based on enhanced chemiluminescence as per the instructions of the manufacturer Quantitative data were obtained using a computing densitometer with a scientific imaging system (Biospectrum AC System, UVP, Upland, CA, USA) Reverse-transcription PCR (RT-PCR) RT-PCR analyses were performed as described previously (Huang et al., 2014) Primers used for amplification of the COX-2 and GAPDH fragments were as follows: COX-2, sense 5′-CCCCCACAGTCAAAGACACT-3′ and antisense 5′-CTCA TCACCCCACTCAGGAT-3′; and GAPDH, sense 5′-CCTTCA TTGACCTCAACTAC-3′ and antisense 5′-GGAAGGCCAT GCCAGTGAGC-3′ GAPDH was used as the internal control The PCR was performed with the following conditions: a denaturation step at 94°C, 30 cycles of a 30 s denaturation step at 94°C, a 30 s annealing step at 56°C, and a 45 s extension step at 72°C to amplify COX-2 and GAPDH cDNA The amplified fragment sizes for COX-2 and GAPDH were 191 and 594 bp respectively PCR products were run on an agarose gel, stained with ethidium bromide, and visualized by UV illumination Measurements of PGE2 production After treatment as indicated, PGE2 in the medium was assayed using a PGE2 enzyme immunoassay kit (Enzo Life Sciences, Butler Pike, PA, USA), according to procedures described by the manufacturer Cell viability assay (trypan blue dye exclusion assay) RAW264.7 macrophages (104 cells per well) were treated with vehicle or WMJ-S-001 for 24 h Cells were harvested and re-suspended in PBS Cells in a volume of 150 μL were mixed BJP with 0.4% trypan blue solution (150 μL) (Sigma-Aldrich, St Louis, MO, USA) From this, 10 μL was charged into the haemocytometer chamber and examined immediately Live cells excluded the dye whereas the dye entered and stained the dead cells blue in colour Both stained and unstained cells were counted, and cell viability was calculated using the formula: cell viability (%) = a/(a + b) × 100, where a is total cells unstained and b is total cells stained Transfection in RAW264.7 macrophages RAW264.7 macrophages were transfected with different constructs as described below using Turbofect reagent (Millipore) according to the manufacturer’s instructions For the reporter assay, cells were transfected with COX-2-luc, NF-κB-Luc, C/EBP-luc, m NF-κB-COX-2-luc, or mC/EBP-COX-2-luc plus Renilla-luc For immunoblotting, cells were transfected with pcDNA, or MKP-1-DN Dual luciferase reporter assay Cells with and without treatments were then harvested, and the luciferase activity was determined using a Dual-Glo luciferase assay system kit (Promega) according to the manufacturer’s instructions Normalization was performed with renilla luciferase activity as the basis Immunofluorescence analysis For determination of NF-κB p65 nuclear translocation, RAW264.7 macrophages were seeded on glass cover slips for 24 h Cells were pretreated with WMJ-S-001 for 30 and stimulated with LPS for another h After treatment, cells were washed twice with PBS and fixed in 4% (v·v−1) paraformaldehyde in PBS for 15 at room temperature Cells were permeabilized for in 0.1% (v·v−1) Triton X-100, and incubated with 5% (v·v−1) skimmed milk in PBS for 30 before being stained To observe NF-κB translocation, cells were reacted with rabbit anti-mouse NF-κB p65 antibody (1:50 dilution in PBS) for h at room temperature After being washed, slides were incubated for h with FITCconjugated goat anti-rabbit IgG and propidium iodide (10 μg·mL−1), and then observed under a confocal microscope (Zeiss, LSM 410) Green fluorescence indicated NF-κB p65, and red fluorescence represented nuclei NF-κB translocation occurred in the cells in which green and red overlapped Chromatin immunoprecipitation (ChIP) assay A ChIP assay was performed as described previously (Huang et al., 2013) Ten per cent of the total purified DNA was used for the PCR in 50 μL reaction mixture The 163 and 173 bp COX-2 promoter fragments between −466 and −395 (for p65 binding sequence), and −138 and −85 (for C/EBPβ binding sequence) were amplified using the primer pairs, for p65 binding sequence, sense: 5′-GTAGCTGTGTGCGTGCTCTG-3′ and antisense: 5′-CTCCGGTTTCCTCCCAGT-3′; for C/EBPβ binding sequence, sense: 5′-AGCTCTCTTGGCACCACC t-3′ and antisense: 5′-ACGTAGTGGTGACTCTGTCTTTCCGC-3′, in 30 cycles of PCR This was done: at 95°C for 30 s, at 56°C for 30 s, and at 72°C for 45 s The PCR products were analysed by 1.5% agarose gel electrophoresis Suppression of MKP-1 expression For MKP-1 suppression, predesigned small interfering (si)RNA targeting the mouse MKP-1 gene were purchased from Sigma British Journal of Pharmacology (2015) 172 1894–1908 1897 BJP W-C Chen et al The siRNA oligonucleotides targeting the coding regions of mouse MKP-1 messenger (m)RNA were as follows: MKP-1 siRNA, 5′-cugguucaacgaggcuauu-3′; a negative control siRNA comprising a 19 bp scrambled sequence with 3′dT overhangs was also purchased from Sigma MKP-1 activity assay A serine/threonine phosphatase assay system (Promega) was used to measure MKP-1 activity according to the manufacturer’s instructions with modifications Briefly, cells were lysed in an extraction buffer Equal amounts of cell homogenates were treated with WMJ-S-001 (1–30 μM) for 20 The reaction mixture were then incubated for h at 4°C with μg anti-MKP-1 antibody (Millipore), and 20 μL protein AMagnetic Beads (Millipore), to immunoprecipitate MKP-1 Immune complexes were then collected, washed three times, and incubated with phosphoprotein, the substrate (amino acid sequence RRApTVA, 100 μM), in protein phosphatase assay buffer (20 mM 4-morpholinepropanesulfonic acid (pH 7.5), 60 mM 2-mercaptoethanol, 0.1 M NaCl, and 0.1 mg mL−1 serum albumin) Reactions were initiated by the addition of the phosphoprotein substrate and carried out for 10 at 30°C We also prepared appropriate phosphate standard solutions containing free phosphate for construction of a standard curve Reactions were terminated by the addition of 50 μL of the Molybdate Dye solution The absorbance at 630 nm was measured on a microplate reader Nonspecific hydrolysis of RRApTVA by lysates was assessed in normal IgG immunoprecipitates Animal model of endotoxaemia To evaluate the effect of WMJ-S-001 on survival rate in endotoxaemic mice, an animal model of endotoxaemia was constructed as described previously (Tsoyi et al., 2011) BALB/c mice, 7–8 weeks-old, were obtained from BioLasco (Taipei, Taiwan) and injected with LPS (15 mg·kg−1, i.p.) to induce endotoxaemia The total number of mice used was 18 After injection of LPS for 12 h, animals were randomized into the vehicle-treated control group and the treatment group, which received WMJ-S-001 20 mg·kg−1 The treatment was administered i.p and repeated 24 h after induction of endotoxaemia Animals were monitored up to 10 days to evaluate survival rate after LPS challenge in the absence or presence of WMJ-S-001 Mice were maintained in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publication 85-23, revised 1996) All protocols were approved by the Taipei Medical University Laboratory Animal Care and Use Committee Statistical analysis Results are presented as the mean ± SEM from at least three independent experiments One-way ANOVA followed by, when appropriate, the Newman–Keuls test was used to determine the statistical significance of the difference between means A P value of < 0.05 was considered statistically significant Reagents LPS purified by phenol extraction from Escherichia coli 0127:B8 was purchased from Sigma-Aldrich (St Louis, MO, USA) U0126, p38 MAPK Inhibitor III, JNK Inhibitor II, 1898 British Journal of Pharmacology (2015) 172 1894–1908 antibodies specific for MKP-1 and COX-2 and Turbofect™ in vitro transfection reagent were purchased from Millipore (Billerica, MA, USA) DMEM, optiMEM, FBS, penicillin and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA) Antibodies specific for C/EBPβ and p65 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) Antibodies specific for α-tubulin, p-p65 Ser536, JNK, p38MAPK, myc tag, and anti-mouse and anti-rabbit IgG-conjugated HRP antibodies were purchased from GeneTex, Inc (Irvine, CA, USA) Antibodies specific for ERK1/2, p-ERK1/2, p-JNK1/2 and p-p38MAPK were purchased from Cell Signaling Technology (Beverly, MA, USA) Murine COX-2 promoter with wild-type construct (−966/+23) cloned into pGL3-basic vector (Promega, Madison, WI, USA) were kindly provided by Dr Byron Wingerd (Michigan State University, East Lansing, MI, USA) The catalytically inactive murine MKP1-C258S [MKP1 dominant-negative mutant (DN) ] were kindly provided by Dr Nicholas Tonks (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) NF-κB-Luc, Renilla-luc and Dual-Glo luciferase assay system were purchased from Promega The C/EBP-luc reporter construct was kindly provided by Dr Kjetil Tasken (University of Oslo, Oslo, Norway) All materials for immunoblotting were purchased from GE Healthcare (Little Chalfont, UK) All other chemicals were obtained from Sigma-Aldrich (St Louis, MO, USA) Results WMJ-S-001 inhibits LPS-induced COX-2 expression To assess the anti-inflammatory activities of aliphatic hydroxamate derivatives, WMJ-S compounds, we evaluated four WMJ-S compounds (Figure 1A) on LPS-induced iNOS and COX-2 expression in RAW264.7 macrophages at the concentration of 10 μM As shown in Figure 1B, WMJ-S-001, -002 and -003 significantly decreased LPS-induced iNOS and COX-2 expression while WMJ-S004 was without effects As WMJ-S-001 exhibited the most marked inhibitory effect, we further investigated the inhibitory mechanisms of WMJ-S001 We examined iNOS and COX-2 levels in RAW264.7 macrophages exposed to various concentrations of WMJ-S001 in the presence of LPS As shown in Figure 1C, WMJ-S001 inhibited LPS-induced iNOS and COX-2 expressions in a concentration-dependent manner In addition, treatment of cells with LPS significantly induced PGE2 formation and COX-2-catalysed PGE2 formation was concentrationdependently inhibited by WMJ-S-001 in LPS-stimulated RAW264.7 macrophages (Figure 1D) We also used a trypan blue dye exclusion test to determine whether the cytotoxic effect was attributable to WMJ-S-001’s inhibitory actions on LPS-stimulated RAW264.7 macrophages As shown in Figure 1E, cell viability was not altered after WMJ-S-001 (1–10 μM) treatment for 24 h compared with the vehicletreated control group Taken together, these findings suggest that WMJ-S-001 may inhibit LPS-induced iNOS and COX-2 expressions in RAW264.7 macrophages Moreover, WMJ-S-001 at concentrations ranging from to 10 μM inhibited LPS-induced COX-2 expression by 60–80% and the IC50 of WMJ-S-001 was approximately 0.3 μM (Supporting Infor- WMJ-S-001 suppresses LPS-induced COX-2 expression mation Fig S1) WMJ-S-001 at the higher concentration (10 μM) did not alter cell viability in RAW264.7 macrophages We thus used WMJ-S-001 at concentrations ranging from to 10 μM to the following experiments WMJ-S-001 suppressed NF-κB and C/EBPβ activation in LPS-stimulated RAW264.7 macrophages The RT-PCR analysis was used to confirm the hypothesis that WMJ-S-001 inhibition of COX-2 expression was attributable to a decrease in cox-2 mRNA As shown in Figure 2A, LPS significantly induced an increase in cox-2 mRNA and WMJS-001 concentration-dependently reduced this effect of LPS Treatment of cells with WMJ-S-001 significantly reduced the LPS-induced increase in COX-2 promoter luciferase activity as determined by the reporter assay (Figure 2B) These results suggest that WMJ-S-001 inhibition of LPS-induced COX-2 expression may have resulted from transcriptional downregulation of cox-2 It is thus conceivable that WMJ-S-001 may suppress the activation of transcription factors that leads to COX-2 expression in LPS-stimulated RAW264.7 macrophages Many consensus sequences, including those for NF-κB and C/EBPβ in the 5′ flanking region of the cox-2 gene, have been reported to up-regulate COX-2 expression in response to various stimuli (Hsu et al., 2011; Chuang et al., 2014) To confirm the regulatory role of NF-κB and C/EBPβ in COX-2 transcription in RAW264.7 macrophages exposed to LPS, the wild-type murine COX-2 reporter construct (−966/ −23, WT COX-2-luc) and mutant reporter constructs with either a NF-κB (−402/−395, mNF-κB COX-2-luc) or a C/EBPβ (−138/−130, m C/EBPβ COX-2-luc) site deletion were separately transfected into RAW264.7 cells As shown in Figure 2C, LPS caused an increase in COX-2 promoter luciferase activity in cells transfected with the wild-type murine COX-2 construct However, LPS-induced COX-2 promoter luciferase activity was reduced in cells transfected with the mutant constructs with NF-κB or C/EBPβ deletion These results suggest that the activation of NF-κB or C/EBPβ is necessary for LPS-induced COX-2 expression in RAW264.7 macrophages We next investigated whether WMJ-S-001 affects NF-κB and C/EBPβ activation in LPS-stimulated RAW264.7 macrophages As shown in Figure 2D, WMJ-S-001 significantly inhibited LPS-induced NF-κB subunit p65 phosphorylation NF-κB/p65 phosphorylation may account for its nuclear localization and transcriptional activity (Li and Verma, 2002) Therefore, we examined whether WMJ-S-001 interferes with the subcellular localization of NF-κB p65 exhibited a decreased cytoplasmic localization and increased nuclear localization after exposure to LPS as determined by immunofluorescence analysis (Figure 2E) WMJ-S-001 markedly impaired the translocation of p65 from the cytosol to nucleus in cells exposed to LPS (Figure 2E) The results from the reporter assays also demonstrated that WMJ-S-001 reduced LPS-increased NF-κB-luciferase activities in RAW264.7 macrophages (Figure 2F) It has been reported that phosphorylation of C/EBPß promotes its transcriptional activity (Nakajima et al., 1993) We therefore determined the phosphorylation status of C/EBPß in RAW264.7 macrophages exposed to LPS As shown in Figure 3A, LPS caused an increase in C/EBPß phosphorylation in a time-dependent manner Treatment of cells with WMJ- BJP S-001 significantly suppressed LPS-induced C/EBPß phosphorylation (Figure 3B) The results from the reporter assays also demonstrated that WMJ-S-001 reduced LPS-increased C/EBPluciferase activities in RAW264.7 macrophages (Figure 3C) We next performed ChIP experiments to determine whether WMJ-S-001 affects the recruitment of p65 and C/EBPß to the endogenous COX-2 promoter region in LPS-stimulated RAW264.7 macrophages The primers encompassing the COX-2 promoter regions between −230 and −60 (containing the C/EBPß-binding site), and between −466 and −304 (containing the NF-κB-binding site) were used As shown in Figure 3D, C/EBPß binding to the COX-2 promoter region (−230/−60) was increased readily after LPS exposure (Figure 3Da) p65 binding to the COX-2 promoter region (−466/−304) was also increased in cells exposed to LPS (Figure 3Db) The COX-2 promoter region (−230/−60 or −466/ −304) was detected in the cross-linked chromatin sample before immunoprecipitation (bottom panels of Figure 3Da and Db, input, positive control) Furthermore, LPS’s effects on C/EBPß and p65 binding to the COX-2 promoter region were reduced in the presence of WMJ-S-001 (Figure 3Ea and Eb) These results suggest that C/EBPß and p65 may be responsible for the inhibitory effects of WMJ-S-001 on COX-2 expression in LPS-stimulated RAW264.7 macrophages p38MAPK signalling contributes to WMJ-S-001 suppression of COX-2 expression in LPS-stimulated RAW264.7 macrophages LPS induction of COX-2 occurs via activation of MAPKs including ERK, JNK and p38MAPK To confirm whether ERK, p38MAPK or JNK1/2 signalling contributes to LPS-induced COX-2 expression in RAW264.7 macrophages, pharmacological inhibitors U0126 (an ERK signalling inhibitor, ERK-I), p38MAPK inhibitor III (a p38MAPK inhibitor, p38-I) and JNK1/2 inhibitor II (a JNK1/2 inhibitor, JNK-I) were used As shown in Figure 4A, these three inhibitors were all effective in reducing LPS-induced COX-2 expression, suggesting the causal role of MAPKs in COX-2 expression in LPS-stimulated RAW264.7 macrophages In addition, these three inhibitors did not alter cell viability as determined by trypan blue dye exclusion assay (Supporting Information Fig S2) It appears that the inhibitory effects of these three inhibitors on LPSinduced COX-2 expression were not attributable to cytotoxic effects We next examined whether WMJ-S-001 affects the status of MAPKs activation to explore the inhibitory mechanisms of WMJ-S-001 on LPS-stimulated RAW264.7 macrophages As shown in Figure 4B, LPS caused increases in ERK, JNK and p38MAPK phosphorylation in RAW264.7 macrophages The increase in ERK (Figure 4C) and JNK (Figure 4D) phosphorylation was not altered in the presence of WMJ-S-001 In contrast, WMJ-S-001 significantly inhibited LPS-induced p38MAPK phosphorylation in RAW264.7 macrophages (Figure 4E) Furthermore, we also used p38MAPK inhibitor III to confirm whether p38MAPK signalling contributes to LPS-induced C/EBPß and p65 phosphorylation As shown in Figure 4F, p38MAPK inhibitor III at μM significantly inhibited LPS-induced C/EBPß phosphorylation Similarly, p38MAPK inhibitor III was also effective in attenuating LPS-induced p65 phosphorylation (Figure 4G) Taken together, these results suggest that WMJ-S-001 may inhibit British Journal of Pharmacology (2015) 172 1894–1908 1899 BJP W-C Chen et al Figure WMJ-S-001 inhibited p65 activation in LPS-stimulated RAW264.7 macrophages (A) Cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another h The extent of COX-2 mRNA was then determined by an RT-PCR assay as described in the ‘Methods’ section Each column represents the mean ± SEM of five independent experiments *P < 0.05, compared with the control group; # P < 0.05, compared with the group treated with LPS alone (B) Cells were transiently transfected with COX-2-luc and renilla-luc for 48 h After transfection, cells were treated with vehicle or WMJ-S-001 (1–10 μM) for 30 min, followed by treatment with LPS (1 μg·mL−1) for another 24 h Luciferase activity was then determined as described in the ‘Methods’ section Data represent the mean ± SEM of at least five independent experiments performed in duplicate *P < 0.05, compared with the control group; #P < 0.05, compared with the group treated with LPS alone (C) Cells were transiently transfected with WT COX-2-luc, mC/EBP-COX-2-luc (C/EBP site mutant) or mNFκB COX-2-luc (NFκB site mutant) and renilla-luc for 48 h Luciferase activity was then determined after treatment with LPS (1 μg·mL−1) for another 24 h Data represent the mean ± SEM of six independent experiments performed in duplicate (D) Cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another 20 The extent of p65 phosphorylation was then determined by immunoblotting Each column represents the mean ± SEM of four independent experiments *P < 0.05, compared with the control group; #P < 0.05, compared with the group treated with LPS alone (E) Cells were pretreated with the vehicle or WMJ-S-001 (10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another h p65 translocation was determined by immunofluorescence analysis as described in the ‘Methods’ section Results shown are representative of at least three independent experiments (F) Cells were transiently transfected with NF-κB-luc and renilla-luc for 48 h After transfection, cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another 24 h Luciferase activity was then determined Data represent the mean ± SEM of six independent experiments performed in duplicate *P < 0.05, compared with the vehicle-treated group; #P < 0.05, compared with the group treated with LPS alone PI, propidium iodide 1900 British Journal of Pharmacology (2015) 172 1894–1908 WMJ-S-001 suppresses LPS-induced COX-2 expression BJP Figure WMJ-S-001 attenuated C/EBPβ activation in LPS-stimulated RAW264.7 macrophages (A) Cells were treated with LPS (1 μg·mL−1) for the indicated time periods Cells were then harvested and C/EBPβ phosphorylation was determined by immunoblotting Each column represents the mean ± SEM of at least five independent experiments *P < 0.05, compared with the control group (B) Cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another 20 The extent of C/EBPβ phosphorylation was then determined by immunoblotting Each column represents the mean ± SEM of five independent experiments *P < 0.05, compared with the control group; #P < 0.05, compared with the group treated with LPS alone (C) Cells were transiently transfected with C/EBP-luc and renilla-luc for 48 h After transfection, cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another 24 h Luciferase activity was then determined Data represent the mean ± SEM of four independent experiments performed in duplicate *P < 0.05, compared with the vehicle-treated group; #P < 0.05, compared with the group treated with LPS alone Cells were treated with LPS (1 μg·mL−1) for the indicated time intervals (D) or treated with WMJ-S-001 (10 μM) for 30 followed by the treatment with LPS (1 μg·mL−1) for another h (E) ChIP assay was performed as described in the ‘Methods’ section Typical traces representative of at least three independent experiments with similar results are shown British Journal of Pharmacology (2015) 172 1894–1908 1901 BJP W-C Chen et al Figure Effects of WMJ-S-001 on p38MAPK signalling cascade in LPS-stimulated RAW264.7 macrophages (A) Cells were pretreated with p38MAPK inhibitor III (p38-I, μM), U0126 (ERK-I, 10 μM) or JNK inhibitor II (JNK-I, 10 μM) for 30 followed by treatment with LPS (1 μg·mL−1) for another 24 h The COX-2 level was then determined by immunoblotting Each column represents the mean ± SEM of at least four independent experiments *P < 0.05, compared with the vehicle-treated group; #P < 0.05, compared with the group treated with LPS alone Cells were pretreated with the vehicle or WMJ-S-001 (1–10 μM) for 30 before treatment with LPS (1 μg·mL−1) for another 20 Figures shown in (B) are representative of at least four independent experiments with similar results The compiled results of ERK (C), JNK (D) or p38MAPK (E) phosphorylations are shown Each column represents the mean ± SEM of at least four independent experiments *P < 0.05, compared with the vehicle-treated group; #P < 0.05, compared with the group treated with LPS alone Cells were pretreated with the vehicle or p38MAPK inhibitor III (p38-I, μM) for 30 before treatment with LPS (1 μg·mL−1) for another 20 The extent of C/EBPβ (F) or p65 (G) phosphorylation was then determined by immunoblotting Each column represents the mean ± SEM of at least four independent experiments *P < 0.05, compared with the vehicle-treated group; #P < 0.05, compared with the group treated with LPS alone 1902 British Journal of Pharmacology (2015) 172 1894–1908 WMJ-S-001 suppresses LPS-induced COX-2 expression p38MAPK signalling to suppress C/EBPß and p65 phosphorylation and COX-2 expression in LPS-stimulated RAW264.7 macrophages MKP-1 plays a causal role in WMJ-S-001 suppression of COX-2 expression in LPS-stimulated RAW264.7 macrophages We next explored the underlying mechanisms of WMJ-S-001 in inhibiting LPS-induced p38MAPK phosphorylation It is likely that WMJ-S-001 may activate a protein phosphatase that dephosphorylates p38MAPK and thereby down-regulates COX-2 expression Several studies have showed that MKP-1 plays a crucial role in regulating inflammatory responses by dephosphorylating and inactivating p38MAPK (Jeffrey et al., 2007; Hsu et al., 2011; Chuang et al., 2014) We thus investigated whether MKP-1 mediates WMJ-S-001 dephosphorylation of p38MAPK in LPS-stimulated RAW264.7 macrophages An MKP-1-DN construct was employed to confirm more specifically that WMJ-S-001’s inhibitory actions on LPS-induced p38MAPK phosphorylation were mediated by MKP-1 As shown in Figure 5A, transfection of cells with MKP-1-DN restored WMJ-S-001-decreased p38MAPK phosphorylation in LPS-stimulated RAW264.7 macrophages The decreases in C/EBP (Figure 5B) and p65 (Figure 5C) phosphorylation by WMJ-S-001 were also prevented by MKP-1-DN Whether MKP-1-DN alters the inhibitory effects of WMJ-S-001 on LPSinduced COX-2 expression was also determined As shown in Figure 5D, transfection of cells with MKP-1-DN significantly restored WMJ-S-001-decreased COX-2 expression in LPSstimulated RAW264.7 macrophages We also used MKP-1 siRNA to confirm that WMJ-S-001’s inhibitory actions on LPS-induced COX-2 expression were mediated by MKP-1 As shown in Figure 5E, transfection of HUVECs with MKP-1 siRNA significantly restored WMJ-S-001-decreased COX-2 expression in RAW264.7 macrophages exposed to LPS Furthermore, WMJ-S-001 caused an increase in MKP-1 activity (Figure 5F) To evaluate the suppressive effect of WMJ-S-001 on systemic inflammation induced by endotoxaemia, mice were injected with LPS (15 mg·kg−1, i.p.) As shown in Figure 5F, WMJ-S-001 at 20 mg·kg−1 prevented LPS-induced mouse death, and the survival rate was about 65% at 20 mg·kg−1 at 72 h Most of the control mice were dead within 48 h and the survival rate was about 10% at 72 h (Figure 5G) Taken together, these results suggest that MKP-1 is responsible for WMJ-S-001-induced suppression of p38MAPK phosphorylation, leading to COX-2-PGE2 down-regulation in LPSstimulated RAW264.7 macrophages Discussion Hydroxamate derivatives exhibit broad pharmacological properties including anti-tumour, anti-infectious and antiinflammatory activities (Jiang et al., 2012; Bertrand et al., 2013; Venugopal et al., 2013; Rodrigues et al., 2014) To develop anti-inflammatory agents for the treatment of dysregulated or excessive inflammatory diseases, we evaluated the effects of a series of novel aliphatic hydroxamate derivatives, WMJ-S compounds, on COX-2 expression in activated macrophages exposed to LPS In this study, we have identified BJP a novel aliphatic hydroxamate derivative, WMJ-S-001, as a potent inhibitor in LPS-stimulated RAW264.7 macrophages The results from the present study demonstrated that WMJS-001 may cause MKP-1 activation to dephosphorylate p38MAPK, leading to the decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated RAW264.7 macrophages.WMJ-S-001 was also shown to protect mice from lethal endotoxaemia induced by LPS in an animal model of endotoxaemia MAPKs are involved in regulating the production of crucial inflammatory mediators such as COX-2 in endothelial cells (Hsu et al., 2011; Chuang et al., 2014) and macrophages (Hsu et al., 2010) It has been reported that p38MAPK and JNK inhibitors exhibit anti-inflammatory activities by suppressing the expression of inflammatory mediators Several novel agents that modulate the JNK and p38 MAPK signalling pathways have been shown to be effective in treating chronic inflammatory diseases (Kumar et al., 2003; Kaminska, 2005) We noted in this study that ERK, JNK and p38MAPK contribute to LPS-induced COX-2 expression in RAW264.7 macrophages However, only p38MAPK signalling blockade was causally related to WMJ-S-001 suppression of COX-2 expression in LPS-stimulated RAW264.7 macrophages Previous studies have demonstrated that MKP-1 negatively regulates p38MAPK, resulting in the down-regulation of proinflammatory mediators (Lasa et al., 2002; Turpeinen et al., 2010) In addition, both p38MAPK and JNK are negatively regulated by MKP-1 in endothelial cells (Hsu et al., 2011; Chuang et al., 2014) MKP-1 was shown to regulate JNK in pulmonary smooth muscle cells (Grant et al., 2007) and p38MAPK in macrophages (Salojin et al., 2006) These observations suggest that the substrate specificity of MKP-1 varies among cell types In addition to p38MAPK and JNK, MKP-1 also dephosphorylates and therefore inactivates ERK (Liu et al., 2005) Furthermore, the MKP-1 phosphatase activity has been reported to be context-dependent In a given situation, not all three MAPKs are targeted for dephosphorylation (Wu and Bennett, 2005a; Wu et al., 2005b) In agreement with these observations, we noted that MKP-1-DN restored WMJ-S-001’s dephosphorylation of p38MAPK It is likely that WMJ-S-001 activates MKP-1 to inactivate p38MAPK signalling, leading to a decrease in the expression of COX-2 in RAW264.7 macrophages Further investigations are needed to explore whether WMJ-S-001 also inactivates p38MAPK signalling through an MKP-1-independent mechanism in RAW264.7 macrophages exposed to LPS The precise mechanism involved in WMJ-S-001-induced MKP-1 activation in RAW264.7 macrophages remains unclear It has been reported that MKP-1 could be regulated through transcriptional and post-transcriptional mechanisms (Liu et al., 2007) In a recent study it was shown that dexamethasone exhibits anti-inflammatory effects by augmenting MKP-1 expression and its activity in LPS-stimulated macrophages (Abraham et al., 2006) We noted that WMJ-S-001 increased MKP-1 activity in RAW264.7 macrophages Further investigations are needed to explore whether WMJ-S-001 augments MKP-1 expression in LPS-stimulated RAW264.7 macrophages In addition, hydroxamate derivatives have been shown to act as histone deacetylase (HDAC) inhibitors to increase acetylation of cellular signalling molecules (Drummond et al., 2005) The acetylation of MKP-1 may British Journal of Pharmacology (2015) 172 1894–1908 1903 BJP W-C Chen et al Figure MKP-1 contributed to WMJ-S-001-induced suppression of p38MAPK, C/EBPβ and p65 phosphorylation and COX-2 expression in RAW264.7 macrophages exposed to LPS Cells were transiently transfected with pcDNA or MKP-1-DN for 48 h followed by 30 of treatment with WMJ-S-001 (10 μM) Cells were then treated with LPS (1 μg·mL−1) for another 24 h (A) or 20 (B–D) The COX-2 level (A) and phosphorylation status of p38MAPK (B), C/EBPβ (C) and p65 (D) were then determined by immunoblotting The compiled results are shown in the bottom of the charts Each column represents the mean ± SEM of at least four independent experiments *P < 0.05, compared with the group treated with LPS alone; #P < 0.05, compared with the LPS-treated group in the presence of WMJ-S-001 (E) Cells were transiently transfected with negative control siRNA or MKP-1 siRNA for 48 h followed by 30 of treatment with WMJ-S-001 (10 μM) Cells were then treated with LPS (1 μg·mL−1) for another 24 h The COX-2 and MKP-1 levels were then determined by immunoblotting The compiled results are shown at the bottom of the charts Each column represents the mean ± SEM of four independent experiments *P < 0.05, compared with the group treated with LPS alone; # P < 0.05, compared with the LPS-treated group in the presence of WMJ-S-001 (F) Cell homogenates were treated with WMJ-S-001 at the indicated concentrations for 20 MKP-1 activity assay was determined as described in the ‘Methods’ section Data represent the mean ± SEM of four independent experiments *P < 0.05, compared with the vehicle-treated control group (G) Mice were pretreated with WMJ-S-001 (20 mg·kg−1) and then stimulated with LPS (15 mg·kg−1, i.p.) in PBS buffer LPS-treated mice with or without WMJ-S-001 treatment were observed for 10 days and their survival rates were recorded Mice in the control group were administered LPS alone (n = 12) and the other group was administered LPS plus 20 mg·kg−1 WMJ-S-001 (n = 6) IP, immunoprecipitation 1904 British Journal of Pharmacology (2015) 172 1894–1908 WMJ-S-001 suppresses LPS-induced COX-2 expression enhance its phosphatase activity (Cao et al., 2008) and increase its interaction with p38MAPK to dephosphorylate p38MAPK (Jeong et al., 2014) WMJ-S-001 was also shown to inhibit HDACs activity (unpubl data) These results raise the possibility that WMJ-S-001 may activate MKP-1 to inhibit COX-2 expression through, at least in part, an inhibitory effect on HDAC Whether WMJ-S-001 increases acetylation of MKP-1 or other signalling molecules, which contribute to its anti-inflammatory effects in RAW264.7 macrophages will need to be investigated further Previous studies reported that transcription factors including NF-κB, C/EBP, SP1 and CREB have been implicated in the induction of COX-2 expression (Kang et al., 2006; Hsu et al., 2010) These transcription factors may participate in a sequential, coordinated regulation of COX-2 expression in LPS-stimulated macrophages (Kang et al., 2006) We noted in reporter and ChIP assays that LPS increased NF-κB and C/EBP-luciferase activities and the recruitment of NF-κB and C/EBPβ to the endogenous cox-2 promoter region in RAW264.7 macrophages This phenomenon was markedly suppressed in the presence of WMJ-S001 The reporter assays further demonstrated that LPSincreased COX-2 promoter luciferase activity was attenuated in cells transfected with the reporter constructs containing a deletion of the NF-κB or the C/EBP site These findings suggest that NF-κB and C/EBPβ play important roles in WMJ-S-001 suppression of LPS-induced COX-2 expression in RAW264.7 macrophages The exact mechanism by which WMJ-S-001 inhibited NF-κB and C/EBPβ binding to the cox-2 promoter region remains to be elucidated Consistent with the previous reports that p38MAPK regulates NF-κB and C/EBPβ (Hsu et al., 2010; Lin et al., 2010), we noted that the p38MAPK inhibitor suppressed LPS-induced NF-κB subunit p65 and C/EBPβ phosphorylation It appears that p38MAPK signalling blockade by MKP-1 may contribute to the inhibitory actions of WMJ-S-001 on NF-κB and C/EBPβ Moreover, we noted in this study that the p38MAPK inhibitor, similar to WMJ-S-001, dose-dependently inhibited p65 phosphorylation C/EBPβ phosphorylation seemed to be similarly inhibited by the p38MAPK inhibitor in a dosedependent manner, and WMJ-S-001 at a lower concentration (1 μM) strongly inhibited C/EBPβ phosphorylation in LPS-stimulated RAW264.7 macrophages Several studies have reported that C/EBPβ phosphorylation is also regulated by ribosomal protein S6 kinase (RSK) (Buck and Chojkier, 2007) and glycogen synthase kinase 3β (GSK3β) (Tang et al., 2005) This raises the possibility that WMJ-S-001 inhibits C/EBPβ phosphorylation through targeting RSK or GSK3β in addition to p38MAPK Further investigations are needed to characterize whether other signalling molecules such as RSK and GSK3β contribute to the anti-inflammatory actions of WMJ-S-001 These observations explain, at least in part, the different patterns of inhibition of C/EBPβ phosphorylation in the presence of the p38MAPK inhibitor and WMJ-S-001 Moreover, physical and functional interactions have been shown to occur between C/EBP and NF-κB family members (Banks and Erickson, 2010) Whether WMJ-S-001 affects the interactions between C/EBPβ and NF-κB or other transcription factors in LPS-stimulated RAW264.7 macrophages needs to be investigated further Furthermore, BJP HDAC3 has been reported to interact with the NF-κB p65 subunit (Chen et al., 2001) and to de-acetylate NF-κB, which contributes to its association with IκBα (Winkler et al., 2012) Hence, it would be worth clarifying whether WMJS-001’s inhibitory actions in LPS-stimulated RAW264.7 macrophages are also attributable to its HDAC inhibitory activity As described earlier, we showed that WMJ-S-001 inhibited LPS-induced COX-2 promoter luciferase activity, suggesting that WMJ-S-001 may modulate COX-2 expression at the transcriptional level However, the control of COX-2 protein expression may also occur at levels other than transcription such as chromatin modifications and post-transcriptional regulation via 3′-UTR (Harper and Tyson-Capper, 2008) There are many adenylate–uridylate-rich elements (AU-rich elements, AREs) and microRNA response elements in the COX-2 3′-UTR When these elements are recognized and bound by specific ARE-binding factors or miRNAs, the stability of COX-2 and its translational efficiency will be affected (Harper and Tyson-Capper, 2008) In addition, the p38MAPK signalling pathways have been shown to contribute to the regulation of COX-2 mRNA stability (Monick et al., 2002) The COX-2 protein stability may also be altered through an N-glycosylation-mediated mechanism or substratedependent degradation process (Mbonye et al., 2008) This raises the possibility that WMJ-S-001 may activate the MKP-1 to suppress COX-2 protein expression by both transcriptional and post-transcriptional or post-translational mechanisms Further investigations are needed to elucidate whether AREbinding factors, miRNAs or protein modifications contribute to WMJ-S-001’s modulation of COX-2 expression in RAW264.7 macrophages In conclusion, we demonstrated in this study that WMJS-001, a novel aliphatic hydroxamate derivative, significantly suppressed COX-2 expression in LPS-stimulated RAW264.7 macrophages We also showed that MKP-1 contributed to the p38MAPK dephosphorylation and subsequent COX-2 downregulation in the presence of WMJ-S-001 Together these findings suggest that WMJ-S-001 may act as a promising therapeutic agent against inflammatory diseases The precise underlying mechanisms of WMJ-S-001 in suppressing inflammation and the more potent compounds derived from its structure should be further characterized and developed Acknowledgements We would like to thank Dr Byron Wingerd (Michigan State University, East Lansing, MI, SUA) for the kind gift of the murine COX-2 promoter with wild-type construct (native −966/+23) and mutant constructs cloned into pGL3-basic vector (Promega); Dr Nichola Tonks (Cold Spring Harbor Laboratory, New York) for the kind gift of the catalytically inactive MKP1-C258S (MKP1-DN); Dr Kjetil Tasken (University of Oslo, Oslo, Norway) for the kind gift of C/EBP-luc reporter construct This work was supported by grant NSC 102-2320-B-038-047 from the Ministry of Science and Technology of Taiwan and grant 102CM-TMU-12 from the Chi Mei Medical Center, Tainan, Taiwan British Journal of Pharmacology (2015) 172 1894–1908 1905 BJP W-C Chen et al Author contributions W.-C C., C.-S Y and M.-J H designed the experiments W C C and C S Y performed the experiments W C C., Y.-F H and M.-J H analysed the data W.-J H contributed reagents and synthesized WMJ-S compounds W.-C C., G O and M.-J H wrote the paper Conflict of interest None Ejima K, Layne MD, Carvajal IM, Kritek PA, Baron RM, Chen YH et al (2003) Cyclooxygenase-2-deficient mice are resistant to endotoxin-induced inflammation and death FASEB J 17: 1325–1327 Farooq A, Zhou MM (2004) Structure and regulation of MAPK phosphatases Cell Signal 16: 769–779 Frazier WJ, Wang X, Wancket LM, Li XA, Meng X, Nelin LD et al (2009) Increased inflammation, impaired bacterial clearance, and metabolic disruption after gram-negative sepsis in Mkp-1-deficient mice J Immunol 183: 7411–7419 Gioannini TL, Weiss JP (2007) Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells Immunol Res 39: 249–260 Glauben R, Batra A, Fedke I, Zeitz M, Lehr HA, Leoni F et al (2006) Histone hyperacetylation is associated with amelioration of experimental colitis in mice J Immunol 176: 5015–5022 References Abraham SM, Lawrence T, Kleiman A, Warden P, Medghalchi M, Tuckermann J et al (2006) Antiinflammatory effects of dexamethasone are partly dependent on induction of dual specificity phosphatase J Exp Med 203: 1883–1889 Alexander SPH, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Spedding M et al (2013) The Concise Guide to PHARMACOLOGY 2013/14: Enzymes Br J Pharmacol 170: 1797–1867 Banks WA, Erickson MA (2010) The blood–brain barrier and immune function and dysfunction Neurobiol Dis 37: 26–32 Bertrand S, Helesbeux JJ, Larcher G, Duval O (2013) Hydroxamate, a key pharmacophore exhibiting a wide range of biological activities Mini Rev Med Chem 13: 1311–1326 Beutler B, Rietschel ET (2003) Innate immune sensing and its roots: the story of endotoxin Nat Rev Immunol 3: 169–176 Buck M, Chojkier M (2007) C/EBPbeta phosphorylation rescues macrophage dysfunction and apoptosis induced by anthrax lethal toxin Am J Physiol Cell Physiol 293: C1788–C1796 Cao W, Bao C, Padalko E, Lowenstein CJ (2008) Acetylation of mitogen-activated protein kinase phosphatase-1 inhibits Toll-like receptor signaling J Exp Med 205: 1491–1503 Chang L, Karin M (2001) Mammalian MAP kinase signalling cascades Nature 410: 37–40 Chen L, Fischle W, Verdin E, Greene WC (2001) Duration of nuclear NF-kappaB action regulated by reversible acetylation Science 293: 1653–1657 Chi H, Barry SP, Roth RJ, Wu JJ, Jones EA, Bennett AM et al (2006) Dynamic regulation of pro- and anti-inflammatory cytokines by MAPK phosphatase (MKP-1) in innate immune responses Proc Natl Acad Sci U S A 103: 2274–2279 Chuang YF, Yang HY, Ko TL, Hsu YF, Sheu JR, Ou G et al (2014) Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells Biochem Pharmacol 88: 372–383 Dickinson RJ, Keyse SM (2006) Diverse physiological functions for dual-specificity MAP kinase phosphatases J Cell Sci 119 (Pt 22): 4607–4615 Grant S, Easley C, Kirkpatrick P (2007) Vorinostat Nat Rev Drug Discov 6: 21–22 Harper KA, Tyson-Capper AJ (2008) Complexity of COX-2 gene regulation Biochem Soc Trans 36 (Pt 3): 543–545 Hotchkiss RS, Karl IE (2003) The pathophysiology and treatment of sepsis N Engl J Med 348: 138–150 Hsu MJ, Chang CK, Chen MC, Chen BC, Ma HP, Hong CY et al (2010) Apoptosis signal-regulating kinase in peptidoglycaninduced COX-2 expression in macrophages J Leukoc Biol 87: 1069–1082 Hsu YF, Sheu JR, Lin CH, Chen WC, Hsiao G, Ou G et al (2011) MAPK phosphatase-1 contributes to trichostatin A inhibition of cyclooxygenase-2 expression in human umbilical vascular endothelial cells exposed to lipopolysaccharide Biochim Biophys Acta 1810: 1160–1169 Huang YH, Yang HY, Hsu YF, Chiu PT, Ou G, Hsu MJ (2013) Src contributes to IL6-induced vascular endothelial growth factor-C expression in lymphatic endothelial cells Angiogenesis 17: 407–418 Huang YH, Yang HY, Hsu YF, Chiu PT, Ou G, Hsu MJ (2014) Src contributes to IL6-induced vascular endothelial growth factor-C expression in lymphatic endothelial cells Angiogenesis 17: 407–418 Jeffrey KL, Camps M, Rommel C, Mackay CR (2007) Targeting dual-specificity phosphatases: manipulating MAP kinase signalling and immune responses Nat Rev Drug Discov 6: 391–403 Jeong Y, Du R, Zhu X, Yin S, Wang J, Cui H et al (2014) Histone deacetylase isoforms regulate innate immune responses by deacetylating mitogen-activated protein kinase phosphatase-1 J Leukoc Biol 95: 651–659 Jiang J, Thyagarajan-Sahu A, Krchnak V, Jedinak A, Sandusky GE, Sliva D (2012) NAHA, a novel hydroxamic acid-derivative, inhibits growth and angiogenesis of breast cancer in vitro and in vivo PLoS ONE 7: e34283 Jin J, Samuvel DJ, Zhang X, Li Y, Lu Z, Lopes-Virella MF et al (2011) Coactivation of TLR4 and TLR2/6 coordinates an additive augmentation on IL-6 gene transcription via p38MAPK pathway in U937 mononuclear cells Mol Immunol 49: 423–432 Dong C, Davis RJ, Flavell RA (2002) MAP kinases in the immune response Annu Rev Immunol 20: 55–72 Kaminska B (2005) MAPK signalling pathways as molecular targets for anti-inflammatory therapy – from molecular mechanisms to therapeutic benefits Biochim Biophys Acta 1754: 253–262 Drummond DC, Noble CO, Kirpotin DB, Guo Z, Scott GK, Benz CC (2005) Clinical development of histone deacetylase inhibitors as anticancer agents Annu Rev Pharmacol Toxicol 45: 495–528 Kang YJ, Wingerd BA, Arakawa T, Smith WL (2006) Cyclooxygenase-2 gene transcription in a macrophage model of inflammation J Immunol 177: 8111–8122 1906 British Journal of Pharmacology (2015) 172 1894–1908 WMJ-S-001 suppresses LPS-induced COX-2 expression Kumar S, Boehm J, Lee JC (2003) p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases Nat Rev Drug Discov 2: 717–726 Lang R, Hammer M, Mages J (2006) DUSP meet immunology: dual specificity MAPK phosphatases in control of the inflammatory response J Immunol 177: 7497–7504 Lasa M, Abraham SM, Boucheron C, Saklatvala J, Clark AR (2002) Dexamethasone causes sustained expression of mitogen-activated protein kinase (MAPK) phosphatase and phosphatase-mediated inhibition of MAPK p38 Mol Cell Biol 22: 7802–7811 Li Q, Verma IM (2002) NF-kappaB regulation in the immune system Nat Rev Immunol 2: 725–734 Lin ML, Lu YC, Chung JG, Wang SG, Lin HT, Kang SE et al (2010) Down-regulation of MMP-2 through the p38 MAPK-NF-kappaB-dependent pathway by aloe-emodin leads to inhibition of nasopharyngeal carcinoma cell invasion Mol Carcinog 49: 783–797 Liu C, Shi Y, Du Y, Ning X, Liu N, Huang D et al (2005) Dual-specificity phosphatase DUSP1 protects overactivation of hypoxia-inducible factor through inactivating ERK MAPK Exp Cell Res 309: 410–418 Liu SF, Malik AB (2006) NF-kappa B activation as a pathological mechanism of septic shock and inflammation Am J Physiol Lung Cell Mol Physiol 290: L622–L645 Liu Y, Shepherd EG, Nelin LD (2007) MAPK phosphatases – regulating the immune response Nat Rev Immunol 7: 202–212 Mbonye UR, Yuan C, Harris CE, Sidhu RS, Song I, Arakawa T et al (2008) Two distinct pathways for cyclooxygenase-2 protein degradation J Biol Chem 283: 8611–8623 Medvedev AE, Kopydlowski KM, Vogel SN (2000) Inhibition of lipopolysaccharide-induced signal transduction in endotoxintolerized mouse macrophages: dysregulation of cytokine, chemokine, and toll-like receptor and gene expression J Immunol 164: 5564–5574 Monick MM, Robeff PK, Butler NS, Flaherty DM, Carter AB, Peterson MW et al (2002) Phosphatidylinositol 3-kinase activity negatively regulates stability of cyclooxygenase mRNA J Biol Chem 277: 32992–33000 Nakajima T, Kinoshita S, Sasagawa T, Sasaki K, Naruto M, Kishimoto T et al (1993) Phosphorylation at threonine-235 by a ras-dependent mitogen-activated protein kinase cascade is essential for transcription factor NF-IL6 Proc Natl Acad Sci U S A 90: 2207–2211 Pawson AJ, Sharman JL, Benson HE, Faccenda E, Alexander SP, Buneman OP et al.; NC-IUPHAR (2014) The IUPHAR/BPS Guide to PHARMACOLOGY: an expert-driven knowledgebase of drug targets and their ligands Nucl Acids Res 42 (Database Issue): D1098–106 Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs Oncogene 26: 3100–3112 Ricciotti E, FitzGerald GA (2011) Prostaglandins and inflammation Arterioscler Thromb Vasc Biol 31: 986–1000 Rodrigues GC, Feijo DF, Bozza MT, Pan P, Vullo D, Parkkila S et al (2014) Design, synthesis, and evaluation of hydroxamic acid derivatives as promising agents for the management of Chagas disease J Med Chem 57: 298–308 Roth RJ, Le AM, Zhang L, Kahn M, Samuel VT, Shulman GI et al (2009) MAPK phosphatase-1 facilitates the loss of oxidative BJP myofibers associated with obesity in mice J Clin Invest 119: 3817–3829 Salojin KV, Owusu IB, Millerchip KA, Potter M, Platt KA, Oravecz T (2006) Essential role of MAPK phosphatase-1 in the negative control of innate immune responses J Immunol 176: 1899–1907 Shishodia S, Koul D, Aggarwal BB (2004) Cyclooxygenase (COX)-2 inhibitor celecoxib abrogates TNF-induced NF-kappa B activation through inhibition of activation of I kappa B alpha kinase and Akt in human non-small cell lung carcinoma: correlation with suppression of COX-2 synthesis J Immunol 173: 2011–2022 Sweet MJ, Hume DA (1996) Endotoxin signal transduction in macrophages J Leukoc Biol 60: 8–26 Tang QQ, Gronborg M, Huang H, Kim JW, Otto TC, Pandey A et al (2005) Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis Proc Natl Acad Sci U S A 102: 9766–9771 Tsoyi K, Jang HJ, Nizamutdinova IT, Kim YM, Lee YS, Kim HJ et al (2011) Metformin inhibits HMGB1 release in LPS-treated RAW 264.7 cells and increases survival rate of endotoxaemic mice Br J Pharmacol 162: 1498–1508 Turpeinen T, Nieminen R, Moilanen E, Korhonen R (2010) Mitogen-activated protein kinase phosphatase-1 negatively regulates the expression of interleukin-6, interleukin-8, and cyclooxygenase-2 in A549 human lung epithelial cells J Pharmacol Exp Ther 333: 310–318 Venugopal B, Baird R, Kristeleit RS, Plummer R, Cowan R, Stewart A et al (2013) A phase I study of quisinostat (JNJ-26481585), an oral hydroxamate histone deacetylase inhibitor with evidence of target modulation and antitumor activity, in patients with advanced solid tumors Clin Cancer Res 19: 4262–4272 Wang Y, Yu C, Pan Y, Li J, Zhang Y, Ye F et al (2011) A novel compound C12 inhibits inflammatory cytokine production and protects from inflammatory injury in vivo PLoS ONE 6: e24377 Williams CS, Mann M, DuBois RN (1999) The role of cyclooxygenases in inflammation, cancer, and development Oncogene 18: 7908–7916 Winkler AR, Nocka KN, Williams CM (2012) Smoke exposure of human macrophages reduces HDAC3 activity, resulting in enhanced inflammatory cytokine production Pulm Pharmacol Ther 25: 286–292 Wu D, Marko M, Claycombe K, Paulson KE, Meydani SN (2003) Ceramide-induced and age-associated increase in macrophage COX-2 expression is mediated through up-regulation of NF-kappa B activity J Biol Chem 278: 10983–10992 Wu JJ, Bennett AM (2005a) Essential role for mitogen-activated protein (MAP) kinase phosphatase-1 in stress-responsive MAP kinase and cell survival signaling J Biol Chem 280: 16461–16466 Wu W, Pew T, Zou M, Pang D, Conzen SD (2005b) Glucocorticoid receptor-induced MAPK phosphatase-1 (MPK-1) expression inhibits paclitaxel-associated MAPK activation and contributes to breast cancer cell survival J Biol Chem 280: 4117–4124 Zhao Q, Wang X, Nelin LD, Yao Y, Matta R, Manson ME et al (2006) MAP kinase phosphatase controls innate immune responses and suppresses endotoxic shock J Exp Med 203: 131–140 British Journal of Pharmacology (2015) 172 1894–1908 1907 BJP W-C Chen et al Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: http://dx.doi.org/10.1111/bph.13040 Figure S1 WMJ-S-001 concentration-dependently suppressed COX-2 expression in RAW264.7 macrophages exposed to LPS (A) Cells were pretreated with the vehicle or WMJ-S-001 at indicated concentrations for 30 before treatment with LPS (1 μg·mL−1) for another 24 h The COX-2 1908 British Journal of Pharmacology (2015) 172 1894–1908 level was then determined by immunoblotting Each column represents the mean ± SEM of six independent experiments *P < 0.05, compared with the control group; (B) based on the results derived from A, the IC50 of WMJ-S-001 was calculated Figure S2 Effects of MAPK inhibitors on cell viability in RAW264.7 macrophages Cells were pretreated with p38MAPK inhibitor III (p38-I, μM), U0126 (ERK-I, 10 μM) or JNK inhibitor II (JNK-I, 10 μM) for 24 h Cell viability was determined using a trypan blue dye exclusion test Each column represents the mean ± SEM of four independent experiments ISSN 0007-1188 (print) ISSN 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promotional purposes, for creating new collective works, or for resale.Special requests should be addressed to: permissionsuk@wiley.com ... glucocorticoids as effectors of the resolution of inflammation Nature reviews Immunology (1): 62 70 1652 British Journal of Pharmacology (2015) 172 1651–1652 BJP British Journal of Pharmacology DOI:10.1111/bph.12898... http://dx.doi.org/10.1111/bph .2015. 172 . issue -7 Abbreviations AnxA2, annexin A2; Plgn, plasminogen; tPA, tissue plasminogen activator 1664 British Journal of Pharmacology (2015) 172 1664–1 676 © 2014 The Authors British Journal. .. pigs against challenge infection Parasitology 132: 67 71 British Journal of Pharmacology (2015) 172 1653–1663 1663 BJP British Journal of Pharmacology Themed Section: Annexins VII Programme REVIEW