
 pla2s play key roles in numerous cellular processes in physiological and pathological conditions by regulating the release of arachidonic acid ( aa ) , a precursor of important lipid mediators .
secretory phospholipases a2 ( spla2s ) constitute a superfamily of enzymes classified into various groups ( ib , iia , iic , iid , iie , iif , iii , v , and x ) on the basis of their source , amino acid sequence , and biochemical characteristics . among them
a group iia asp49 spla2 , named myotoxin - iii ( mt - iii ) , isolated from bothrops asper snake venom , has been shown to promote marked local inflammatory events in several experimental models [ 47 ] .
some of these events are induced by inflammatory mediators , such as eicosanoids , produced by inflammatory cells .
in addition , we previously showed that this spla2 is capable of inducing cyclooxygenase-2 ( cox-2 ) protein expression and stimulating aa and prostaglandin ( pg)d2 , pge2 production , when incubated with macrophages in culture . despite the importance of prostanoids in the regulation of inflammatory events induced by spla2s , and the relevance of macrophages in this response , the signal transduction pathways that lead to mt - iii - promoted biosynthesis of pgs and cox-2 expression in macrophages are unknown .
cox-1 is present in most tissues   and is responsible for generating pgs for diverse physiological and pathological functions .
cox-2 , in turn , can be constitutively expressed in some tissues but , normally , is inducible under inflammatory conditions in several types of cells [ 1114 ] .
the promoter region of the cox-2 gene contains several binding sites for transcription factors including nf-b , creb , c / ebp , and ap-1 [ 13 , 15 , 16 ] . of these
, nf-b is the main transcription factor involved in cox-2 gene expression in macrophages during inflammatory processes [ 17 , 18 ] .
the involvement of nf-b in cox-2 expression and pge2 production induced by group iia asp49spla2s is unknown , stressing the need to perform studies on this matter .
furthermore , it has been demonstrated that cox-2 expression correlates with the activities of intracellular signaling proteins such as p38 mitogen - activated protein kinase ( p38mapk ) [ 19 , 20 ] , phosphoinositide 3-kinase ( pi3k ) [ 21 , 22 ] , and protein kinase c ( pkc ) [ 21 , 23 ] in macrophages activated by several stimuli . however , the roles of these kinases in group iia spla2-induced cox-2 expression have not been yet investigated in macrophages . since the production of lipid mediators is highly regulated by a variety of extracellular stimuli , it is relevant to study how the spla2s target their action to generate pgs , especially with regard to the expression of cox-2 , a major isoform responsible for the production of pge2 in inflammatory conditions . in this
study the mechanisms by which the spla2mt - iii activates macrophages leading to expression of cox-2 and release of pge2 were investigated , with focus on the involvement of nf-b and the signaling pathways proteins p38mapk , pi3k , and pkc .
pge2 enzyme immunoassay kits and rabbit polyclonal anti - murine cox-2 antibodies were purchased from cayman chemical ( ann arbor , mi , usa ) ; mouse monoclonal anti - rat -actin antibody was from sigma aldrich co. ( st . louis , mo , usa ) ; peroxidase - conjugated secondary sheep anti - mouse or donkey anti - rabbit antibodies were from ge healthcare ( buckinghamshire , uk ) .
sn50 , sb202190 , ly294002 , h7-dihydro were purchased from calbiochem - novabiochem ( la jolla , ca , usa ) .
antibodies against phospho - p38mapk , p38mapk , phospho - pi3k , pi3k , and phospho - pkc were from cell signaling technology ( danvers , ma ) .
the salts used were purchased from merck , ge healthcare and bio - rad ( hercules , ca ) .
animals were housed in temperature - controlled rooms , with a relative humidity of 65.3  0.9% and 12  h dark - light period , and received water and food ad libitum .
the animals and research protocols used in this study followed the guidelines of the ethical committee for use of animals of instituto butantan , sp , brazil ( ceuaib , protocol number 592/09 ) , and international policies of experimental animal care . all efforts were made to minimize the number of animals used and their suffering .
mt - iii was isolated from bothrops asper venom by ion - exchange chromatography on cm - sephadex c-25 using the conditions described by lomonte and gutirrez , followed by rp - hplc on a c8 semipreparative column ( 10  250  mm ; vydac ) eluted at 2.0  ml / min with a 070% acetonitrile gradient containing 0.1% ( v / v ) trifluoroacetic acid , during 30  min , on an agilent 1200 instrument monitored at 215  nm .
homogeneity of the final preparation was assessed by analytical rp - hplc on a c4 column ( 4.6  150  mm ) using a 060% acetonitrile gradient .
the absence of endotoxin contamination in the mt - iii preparation was demonstrated by the quantitative limulus amebocyte lysate ( lal ) test , which revealed undetectable levels of endotoxin ( < 0.125  eu / ml ) .
resident peritoneal macrophages were harvested by washing the peritoneal cavity with 2  ml of apyrogenic saline solution .
aliquots of the washes were used to count total cell numbers in a neubauer chamber after dilution ( 1  :  20 , v / v ) in turk 's solution .
for adhesion , aliquots of either 1  10 or 3  10 cells / ml were added to 24- and 6-well polystyrene culture plates , respectively , and incubated for 3  h , in rpmi 1640 medium supplemented with 1% of l - glutamine and 100  g / ml of garamicine , at 37c and 5% co2 atmosphere .
nonadherent cells were removed by vigorous washing three times with glutamine - free rpmi 1640 . by this procedure ,
peritoneal cells , which were initially composed of 4050% of f4/80 positive cells and more than 30% of cd19 positive cells , became enriched in f4/80 positive cells ( more than 90% of the adhered cells ) .
this concentration was previously shown as noncytototoxic but stimulatory of macrophages functions [ 5 , 8 , 26 ] . at selected time intervals ( 0.5 , 1 , and 4.5  h )
, the plates were centrifuged at 500  g for 6  min at 22c .
the predominance of macrophages , constituting more than 95% of cells in the washes , was confirmed by light microscopic analysis of smears stained with hema ( fisher scientific company , middletown , va ) .
where appropriate , the following inhibitors were used : 2.5  m tpck ( n--tosyl - l - phenylalanine chloromethyl ketone arachidonyl trifluoromethyl ketone ) and 50  g / ml of sn50 , selective inhibitors of nf-b activation ; 1  m sb202190 , an inhibitor of p38mapk ; 25  m ly294002 , an inhibitor of pi3k ; 20  m h7-dihydro , an inhibitor of pkc .
all the above inhibitors were added 60  min before stimulation of macrophages with mt - iii or rpmi ( control ) .
cells treated with either inhibitors or mt - iii or both were analyzed for viability by the tetrazolium - based ( mtt ) colorimetric assay .
no significant changes in cell viability were registered with any of the above agents or the vehicle at the concentrations used ( data not shown ) .
the extraction of the prostaglandin was performed on sep - pak c18 columns ( waters corporation , milford , ma ) and eluted with ethanol . in brief , 50  l aliquots of each extracted sample were incubated with the eicosanoids conjugated with acetylcholinesterase and the specific rabbit antiserum in 96-well plates were coated with anti - rabbit igg mouse monoclonal antibody .
after addition of the substrate , the absorbance of the samples was recorded at 405  nm in a microplate reader ( labsystems multiskan ) , and concentrations of pge2 were estimated from standard curves .
aliquots of 1  10 cells were lysed with 100  l of sample buffer ( 0.5  m tris - hcl , ph 6.8 , 20% sds , 1% glycerol , 1  m -mercaptoethanol , and 0.1% bromophenol blue ) and boiled for 10  min .
the samples were subjected to sds - polyacrylamide gel electrophoresis ( sds - page ) on 10% bisacrylamide gels overlaid with a 5% stacking gel .
proteins were then transferred to nitrocellulose membrane ( ge healthcare , buckinghamshire , uk ) using a mini trans - blot ( bio - rad laboratories , richmond , ca , usa ) .
the membrane was blocked for 1  h with 5% w / v nonfat dry milk in tris - buffered saline - tween 20 ( ttbs ) ( 20  mm  tris , 100  mm  nacl , and 0.5% tween 20 ) and incubated with primary antibodies against cox-2 , cox-1 ( 1  :  1500 and 1  :  500 , resp . ) , and -actin ( 1  :  2000 ) . for the study of expression and activation of protein kinases by mt - iii ,
the membrane was blocked for 1  h in 5% w / v bsa in ttbs and incubated with antibodies against either phospho - p38mapk , p38mapk , phospho - pi3k , pi3k , or phospho - pkc , and pkc at 4c with gentle shaking , overnight .
the membrane was then washed and incubated with appropriate secondary antibody conjugated to horseradish peroxidase .
detection was accomplished using the enhanced chemiluminescence method according to instructions of the manufacturer ( ge healthcare , buckinghamshire , uk ) .
densities of the bands were determined by a gs  800 densitometer ( bio - rad laboratories , richmond , ca ) using the image analysis software quantity one ( bio - rad laboratories , richmond , ca ) .
nuclear extracts from peritoneal adherent cells ( 3  10 cells / well ) were obtained as previously described , and protein concentration was determined according to the bradford method .
nf-b binding capacity was evaluated as previously described . briefly , end - labeled [ -32p ] atp oligonucleotides containing an nf-b consensus - binding site ( 5-agttgaggggactttcccaggc-3 ) were incubated for 20  min at room temperature with 5  g of nuclear extract protein .
dna - protein complexes were then separated on a 5.5% nondenaturing polyacrylamide gel using a running buffer of 45  mm tris , 45  mm borate , and 1  mm edta buffer . the gels were vacuum - dried ( 80c ) and subjected to autoradiography .
the blots were analyzed by scanner densitometry ( storm 840 , dynamic molecular , sunnyvale , ca , usa ) .
differences among groups were analyzed by one - way analysis of variance ( anova ) followed by tukey 's test or by the student 's t - test .
initially , it was verified whether mt - iii induces activation of nf-b in peritoneal isolated macrophages . as demonstrated in figures 1(a ) and 1(b ) , a rapid activation of nf-b was induced by mt - iii since a marked nuclear activation was detected at 30  min of incubation as compared with control cells .
this is a rapid event since after one hour of incubation with mt - iii , neither nf-b activation nor dna binding was observed ( figures 1(c ) and 1(d ) ) .
the participation of the transcription factor nf-b on cox-2 protein expression and pge2 production induced by mt - iii was investigated using specific inhibitors of this pathway .
the compound tpck , which prevents the activation of nf-b , abrogated both cox-2 expression ( figures 2(a ) and 2(b ) ) and pge2 production ( figure 2(e ) ) in mt - iii - stimulated macrophages .
pretreatment of cells with sn50 , a cell permeable peptide that competes specifically with nf-b subunit p50 for the translocation from the cytosol into the nucleus , significantly reduced mt - iii - induced cox-2 protein expression ( figures 2(c ) and 2(d ) ) and pge2 production ( figure 2(e ) ) in resident macrophages by 50% and 30% , respectively . taken together , our data demonstrate that nf-b is involved in both cox-2 protein expression and pge2 production induced by mt - iii in isolated macrophages .
we next verified whether mt - iii causes phosphorylation in kinases that activate important signaling pathways for macrophages function .
as shown in figures 3(a ) , 3(d ) , and 3(g ) , unstimulated macrophages showed a basal phosphorylation on all kinases investigated .
treatment of isolated macrophages with 0.4  m mt - iii resulted in a 3- to 5-fold time - dependent increase in p38mapk , pi3k , and pkc phosphorylation over the corresponding control cells p38 ( figures 3(a ) and 3(b ) ) and pi3k ( figures 3(d ) and 3(e ) ) phosphorylation was detectable as early as one min and was sustained at least for 15  min .
pkc phosphorylation was also detectable and peaked at one min and remained detectable at 15  min after mt - iii addition ( figures 3(g ) and 3(h ) ) .
altogether , the above data demonstrate that mt - iii rapidly activates phosphorylation of protein kinases in macrophages , without altering total p38 , pi3k , and pkc ( figures 3(c ) , 3(f ) , and 3(i ) ) .
it has been previously reported that protein kinases participate in the signaling under group iia spla2s stimuli [ 31 , 32 ] . to assess the role of kinases in the described actions of mt - iii , we determined the effects of the specific inhibitors of p38mapk , pi3k , and pkc ( sb202190 , ly294002 , and h7-dihydro , resp . ) on mt - iii - stimulated pge2 release in macrophages .
mt - iii - induced cox-2 protein expression ( figures 4(a ) and 4(b ) ) and increments in pge2 ( figure 4(c ) ) by macrophages were inhibited by sb202190 , ly294002 , and h7-dihydro when compared to macrophages after 4.5  h of treatment with mt - iii and pretreated with vehicle .
unstimulated macrophages showed a weak basal cox-2 protein expression when pretreated with or without inhibitors of kinases ( figures 4(a ) and 4(b ) ) .
we have previously shown that resident macrophages have enhanced mt - iii - induced cox-2 and pge2 production dependent on nf-b activation .
next , to determine whether the activation of this transcription factor is mediated by kinase signaling pathways , we tested the effect of specific inhibitors of kinases on nf-b activation by mt - iii .
pretreatment of resident macrophages with sb202190 and h7-dihydro , but not ly294002 , completely inhibited mt - iii - induced activation of nf-b . taken together , these results suggest that mt - iii - stimulated cox-2 expression , pge2 synthesi , s and nf-b activation are mediated through the activation of distinct protein kinases , such as p38mapk and pkc pathways .
in this study we examined the effect of the asp49 spla2 mt - iii , isolated from bothrops asper snake venom , on macrophage activation and the mechanisms through which it stimulates cox-2 expression and pge2 production .
several lines of evidence clearly established that nf-b regulates the expression of several inflammatory mediators and enzymes .
we also show that this pathway is important for cox-2 expression and pge2 release in response to this toxin since incubation of macrophages with the inhibitor of ib phosphorylation ( tpck ) blocked mt - iii - induced cox-2 expression and pge2 release .
the involvement of nf-b as the mechanism underlying mt - iii - induced upregulation of cox-2 expression was further confirmed by results with inhibition of nf-b nuclear translocation site by the compound sn50 , which markedly reduced mt - iii - induced cox-2 expression and pge2 synthesis .
thus , mt - iii activates downstream pathways required for upregulation of cox-2 expression through activation of nf-b .
our data are in agreement with findings that a recombinant group iia spla2 induced the activation of nf-b in the macrophage cell line raw 264.7 . to our knowledge , this is the first demonstration of the existence of a link between nf-b and a group iia spla2 leading to expression of cox-2 and production of pge2 . despite various efforts to study in detail the inflammatory mechanisms triggered by group iia asp49 spla2 , the signal transduction mechanism is still unclear . in particular , it is not well understood how the signal transduction pathways are started by extracellular mt - iii stimuli in peritoneal macrophages , since no receptors or acceptors of group iia snake venom spla2 have been described . since protein kinases are part of the signal transduction pathways which connect inflammatory and other extracellular signals with intracellular responses , such as protein synthesis , we investigated the role of some protein kinases which have been shown to participate in cox-2 upregulation induced by inflammatory and infectious stimuli , such as pkc [ 21 , 23 ] , p38mapks , and pi3k [ 21 , 22 ] .
our data demonstrate , for the first time , that a type iia asp49 spla2 from snake venom is able to activate phosphorylation of these kinase proteins in isolated macrophages .
next , by using pharmacological approaches , we investigated the role of these kinases in pge2 release and cox-2 expression .
it was found that mt - iii effects in macrophages are regulated by specific signaling pathways and that the signaling proteins p38mapk and pkc are distinctly involved in cox-2 expression , pge2 release , and activation of nf-b .
our data are consistent with other studies in which activation of p38mapk is a critical link in inflammation , cytotoxicity , and lipid body formation induced by type iia spla2 from both human   and snake venoms [ 26 , 35 ] . in this context
, some works confirmed that p38mapk - nf-b pathway is an important component of cellular signal transduction , especially in regulating inflammatory genes [ 36 , 37 ] and that p38mapk specific inhibitors greatly attenuate nf-b nuclear translocation [ 38 , 39 ] , cox-2 expression , and pge2 release .
similarly , the observation that production of pge2 and expression of cox-2 via nf-b in murine macrophages activated by mt - iii is dependent on the pkc pathway agrees with other studies in that the groups iia and vspla2 activate pkc signaling protein in some cell types [ 41 , 42 ]
. it also agrees with the observation that this protein kinase is required for pge2 biosynthesis , cox-2 expression , and nf-b activation in both raw 264.7 cells and mouse peritoneal macrophages upon inflammatory stimuli [ 43 , 44 ] .
in contrast , we showed that blockade of mt - iii function with pi3k inhibitor is sufficient to suppress both pge2 production and cox-2 expression but is unable to suppress nf-b activation .
the observation that pi3k is critically involved in mt - iii - induced cox-2 and pge2 production is consistent with previous reports that pi3k pathway is recruited for cox-2 expression under different inflammatory conditions [ 45 , 46 ] . since the effect of mt - iii on cox-2 expression and pge2 release
could be explained by an upregulation of b - dependent transcription in murine macrophages , we hypothesize that some of the signaling pathways activated by mt - iii are also exerted through another regulatory element(s ) , because this spla2 still induces the activation of nf-b in the presence of pi3k inhibitor .
it is suggested that , besides nf-b , mt - iii leads to the activation of other types of transcription factors . in agreement with this hypothesis ,
there are reports that pi3k is required for camp response element - binding ( creb ) or activator protein-1 ( ap-1 ) activation by different stimuli for downstream cox-2 protein synthesis [ 47 , 48 ] . although our results have identified selected downstream pathways regulating key steps involved in the biosynthesis of cox-2 expression and pge2 synthesis induced by mt - iii , the mechanism of spla2-iia - mediated pi3k and other protein kinases activation involved in cox-2 upregulation , remains to be determined .
the involvement of distinct pathways mediated by p38mapk / nf-b and pkc / nf-b is essential for mt - iii - induced pge2 release via cox-2 protein .
moreover , our results indicate that there is no crosstalk between pi3k phosphorylation and nf-b activation implicated in mt - iii cox-2 expression and pge2 production in our experimental conditions . taken together ,
the results presented provide new insights into the mechanisms involved in the production of pge2 through the cox-2 pathway by further defining distinct signaling pathways induced by an asp49 iia spla2 from snake venom .
 phospholipases a2 ( pla2 ) are key enzymes for production of lipid mediators . 
 we previously demonstrated that a snake venom spla2 named mt - iii leads to prostaglandin ( pg)e2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 ( cox-2 ) . 
 herein , we explored the molecular mechanisms and signaling pathways leading to these mt - iii - induced effects . 
 results demonstrated that mt - iii induced activation of the transcription factor nf-b in isolated macrophages . by using nf-b selective inhibitors , the involvement of this factor in mt - iii - induced cox-2 expression and pge2 production 
 was demonstrated . 
 moreover , mt - iii - induced cox-2 protein expression and pge2 release were attenuated by pretreatment of macrophages with sb202190 , and ly294002 , and h-7-dihydro compounds , indicating the involvement of p38mapk , pi3k , and pkc pathways , respectively . 
 consistent with this , mt - iii triggered early phosphorylation of p38mapk , pi3k , and pkc . furthermore , sb202190 , h-7-dihydro , but not ly294002 treatment , abrogated activation of nf-b induced by mt - iii . altogether , these results show for the first time that the induction of cox-2 protein expression and pge2 release , which occur via nf-b activation induced by the spla2-mt - iii in macrophages , are modulated by p38mapk and pkc , but not by pi3k signaling proteins . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions 