5-lipoxygenase ( 5-lox ; ec 1.13.11.34 ; encoded by the alox5 gene ) is the key enzyme in the biosynthesis of lipid signaling molecules , that is , the inflammatory leukotrienes and the anti - inflammatory   lipoxins [ 1 , 2 ] . the 5-lox system is considered one of the key players in inflammation , an important pathway in the cardiovascular system ( particularly in atherosclerosis ) [ 4 , 5 ] , and a putative modulator of central nervous system ( cns ) functioning and pathobiology [ 68 ] .
moreover , the 5-lox pathway has been considered in mechanisms of neural plasticity ranging from neurogenesis and neural differentiation   to regulation of synaptic plasticity [ 10 , 11 ] .
for example , metabolites derived from 5-lox activity have been implicated in the expression of long - term depression in hippocampal slices .         
it is believed that a nonleukocyte synthesis of leukotrienes does not require 5-lox and that in the cns and in the heart , leukotrienes can be produced via a transcellular biosynthetic pathway [ 12 , 13 ] .
nevertheless , there is evidence for 5-lox expression in these organs / systems [ 6 , 1416 ] .
thus , regulation of the brain expression of 5-lox participates in mechanisms of neuroprotection . in the cns of rats and mice , 5-lox expression increases during aging [ 14 , 17 ] and in brain ischemia
is elevated in alzheimer 's disease [ 7 , 20 ] and in response to ischemia . in the heart ,
5-lox content appears to remain stable during cardiac remodeling   but can be altered by dietary modifications .
furthermore , in the heart , that is , myocardial cells , drugs such as statins and thiazolidinediones trigger anti - inflammatory processes and increase the production of anti - inflammatory lipoxins depending on 5-lox , particularly the status of 5-lox phosphorylation [ 15 , 23 ] .        
5-lox gene expression is regulated by epigenetic mechanisms including modifications of dna methylation [ 1 , 2426 ] .
epigenetics encompasses the modification of chromatin structure that leads to regulation of the gene expression and phenotype .
epigenetic modifications are further influenced by the environment (  life experience  ) and are typically long lasting and sometimes heritable .
thus , dna methylation at the sites of cpg dinucleotides has been implicated in gene regulation in virtually all tissues , including the brain .
siegmund et al .   studied human cerebral cortex samples in aging subjects and found a progressive aging - associated rise in the dna methylation of 5 cpg islands of certain cns genes , typically in conjunction with declining levels of their corresponding mrnas .
these authors concluded that dna methylation is dynamically regulated in the human cerebral cortex throughout the lifespan , involves differentiated neurons , and affects a substantial portion of genes predominantly by an age - related increase .
in addition , data are available on epigenetic mechanisms and cardiovascular gene regulation . for example
, it was observed that the genomic dna methylation in patients with coronary artery disease is significantly higher than in controls .           
it has been hypothesized that the 5-lox pathway may be involved in the comorbidity of certain heart and brain disorders [ 30 , 31 ] .
furthermore , it was proposed that comparative studies of heart and brain tissues from the same subjects would improve efforts in this area of research . here , we investigated whether aging affects brain and heart 5-lox mrna levels and dna methylation .
because mice are used in many 5-lox - related experimental models , including 5-lox - deficient mice , we investigated mouse tissue samples .
however , a conundrum of epigenetic research of cns- and heart - expressed genes lies not only in the cell - specificity of epigenetic mechanisms , but also in their species - specificity .
thus , the human 5-lox gene is on chromosome 10 , whereas in the mouse it is on chromosome 6 . comparing the dna regions of the 5-lox promoter and exon 1 ( 1844  nt , human ; 1937  nt , mouse ) , we found 132 cpg dinucleotides in the human and only 59 in the mouse gene promoter regions . to study the methylation status of this region , we employed our recently developed methylation - sensitive restriction endonuclease assay .
since dna methylation depends on dna - methyltransferases ( dnmts ) , we investigate the tissue content of mrnas for dnmt1 and dnmt3a , the maintenance , and the de novo dnmts , respectively .
two - month - old ( young ) and 22-month - old ( old ) c57bl/6j male mice were obtained from the national institute on aging ( bethesda , md ) .
they were housed in groups of 46 in a temperature controlled room and had free access to laboratory chow and water .
cerebellum , frontal cortex , hippocampus , and heart ( the apex portion was selected as a source of uniform ventricular myocytes ) tissues were obtained following lethal anesthesia ( 160  mg / kg ketamine ; sigma , st .
louis , mo ) and transcardial perfusion with 0.9% ice - cold saline to remove the circulating blood cells ( i.e. , until the outflow from the right atrium was clear ) .
the total rna was extracted using the chemical isolation method with trizol reagent ( invitrogen , carlsbad , ca ) following the manufacturer 's instructions . to eliminate possible dna contamination ,
rna samples were treated with a dnase reagent , dna - free ( ambion , inc . , austin , tx ) .
total rna ( 3  g ) was reverse transcribed with 200  u of cloned moloney murine leukemia virus ( m - mlv ) reverse transcriptase ( gibco brl , carlsbad , ca ) .
the qpcr was performed in a stratagene mx3005p qpcr system ( stratagene , la jolla , ca ) using maxima sybr green qpcr master mix ( fermentas , glen burnie , md ) in a two - step cycling protocol as described by the manufacturer .
there was an initial 5 minutes denaturing followed by 40 cycles of denaturing at 95c for 15 seconds and annealing / elongation at 60c for 1 minute .
table 1 shows primer sequences used to detect 5-lox , dnmts , and cyclophilin mrnas ( primers were purchased from integrated dna technologies , inc .
, the amplification products of all above - noted genes were run on agarose gels . for each product
, we obtained one single band of the expected size ( data not shown ) .
reactions were performed using three different concentrations ( 12.5 , 25 , 50  ng ) of reverse transcribed material ( in duplicate for each sample ) .
they were homogenized in homogenizing buffer ( 100  mm nacl , 10  mm tris - hcl ph 8.0 , 25  mm edta , 0.5% sds , 0.1  mg / ml proteinase k ) , 100  l per 10  mg of tissue , and incubated 4 - 5 hours at 37c .
the dna was purified with phenol , phenol - chloroform ( ph 8.0 ) , precipitated with isopropanol , and dissolved in h2o .
the dna methylation assay was designed as follows : the promoter - exon - intron structure of the mus musculus 5-lox gene was reconstructed by aligning the 5-lox ( alox5 ) mrna ( nm_009662 ) , the promoter , the 5 untranslated region ( utr ) , and partial coding region sequences ( af393814 ) with the mouse genome sequence .
although the mouse 5-lox gene promoter does not contain typical cpg islands , the mouse 5-lox promoter-5utr - first exon , the adjacent intron portion , and the 3 end of the gene contain the highest c and g densities .
the 5utr - first exon and the adjacent intron region ( comprising 519 nucleotides ) contain 66% of all c and g nucleotides .
furthermore , this region contains 41  cpg dinucleotides ; 14 are located upstream from the atg translation start codon and 27 are downstream ( figure 1 ) .
multiple methylation - sensitive endonuclease recognition sites were located in that portion . thus , we investigated their methylation rate ( i.e. , upstream and downstream from the atg translation start codon ) with the aid of four methylation - sensitive endonucleases  acii ( c  cgc ) , bstui ( cg  cg ) , hinp1i ( g  cgc ) , and hpaii ( c  cgg ) ( their respective recognition sequences cutting sites are shown in parentheses ) . to measure the 5-lox dna methylation levels
, we used the restriction digest - quantitative pcr ( sybr green rd - qpcr ) .
the selected approach utilizes the ability of methylation - sensitive endonucleases to digest only unmethylated recognition sites , and their inability to act on sites with methylated cytosine .
thus , if the targeted * cpg is methylated , the site is blocked for the enzyme 's endonuclease activity and as a result , greater amounts of templates are available for the action of the taq dna polymerase .
the assay was performed as follows : 1  g of genomic dna was used in a restriction digest reaction for each of the four endonucleases . digested dna samples were diluted with water and an aliquot ( 100  ng dna )
was used for qpcr ( stratagene ) with the maxima sybr green qpcr master mix ( fermentas ) according to the manufacturer 's protocol .
the following primers were used : forward 5-agagaaggatgcgttggaaggt-3 and reverse 5-gactccgggcaagtgagtgct -3. these primers amplify the 238  nt region upstream of the first atg translation start codon .
this region contains two recognition sites for each of 4 endonucleases selected for the assay .
primers for the exon - intron part were as follows : forward 5-agtcatgccctcctacacggtca-3 , reverse 5-agtcatgccctcctacacggtca-3. they amplify a 344  bp fragment . for the input control , we used the 394  nt region in the first intron because this region does not contain recognition sites for the selected methylation - sensitive endonucleases .
this region was amplified with the following primers : forward 5-tgatgtggctggcctcttatgtga-3 ,   reverse 5-actgggactgagtgcaggaaatgt-3. the qpcr reactions were run with 2 different primer sets ( target and input ) in separate tubes and the coefficient of variation for the relative amount of a target sequence was calculated .
data were analyzed by either one - way anova followed by dunnett 's multiple comparison test , or by an independent sample t - test .
results are expressed as the mean    s.e.m . from 5 to 10 animals .

 figure 3 shows that the expression levels of 5-lox mrna in heart tissue are about fifty times greater than in brain tissue .
similar to previously published data , we found that during aging , 5-lox content in the brain ( e.g. , cerebellum and frontal cortex ) increases .
however , in the heart , aging resulted in significantly decreased 5-lox mrna levels ( figure 3 ) .           using the 5-lox dna methylation assay , which is based on the ability of methylation - sensitive endonucleases to digest only unmethylated recognition sites
, we found that the four endonucleases  acii , bstui , hinp1i , and hpaii  revealed a differential tissue- and brain - region - specific methylation status of 5-lox dna ( figure 4 ) .
the dna region targeted by acii was more methylated in the heart compared to the brain ; whereas the dna regions targeted by bstui , hinp1i , and hpaii were more methylated in the brain ( frontal cortex and hippocampus but not the cerebellum ) than in the heart ( figure 4 )
. aging generally increased 5-lox dna methylation but this effect was differential with respect to the dna region studied , the type of the tissue / organ ( e.g. , heart versus brain ) , and the brain region we investigated . focusing on the promoter-5utr region ( figure 4 ) , we found the effect of aging in the areas targeted by acii , bstui , and hpaii but not hinp1l .
thus , the methylation levels assayed by acii and bstui were increased by aging in all tissues studied , whereas the methylation levels studied by hpaii were increased only in the hippocampus .
focusing on the exon - intron region , we found that aging increased methylation only in the brain and only in the dna region sensitive to bstui ( units as in figure 4 ( two - tail t - test ) ; frontal cortex : young = 110      14 , old = 163  14 ,   p < .05 , n = 10 ; hippocampus : young = 86  8 , old = 136  17 , p < .05 , n = 5 ; cerebellum : young = 36  6 , old = 58  10 , n.s . ,
n = 5 ; heart : young = 42  20 , old = 54  10 ; ns , n = 5 ) .       
the content of mrna for dnmt1 and dnmt3a ( maintenance and the de novo dnmts , resp . ) was generally greater in the heart compared to the brain , particularly the content of dnmt3a mrna ( figure 5 ) .
the only aging - associated difference we observed was the decreased dnmt3a mrna content in the heart of old versus young mice ( figure 5(b ) ) .
in line with previous reports , we confirmed that 5-lox mrna is expressed in the mouse brain , albeit at levels significantly lower than 5-lox mrna levels measured in heart tissue .
we also confirmed that aging increases 5-lox mrna content in the brain . in our previous work with rat brain tissue
, we found an aging - associated 5-lox increase in the cerebellum and the hippocampus . in this study with mice
, we found an aging - associated 5-lox increase in the cerebellum and the frontal cortex but not in the hippocampus .
these differences do not appear to be species - related ( e.g. , rat versus mouse ) because others found that aging increases 5-lox expression in the mouse hippocampus but not in the cerebellum and cortex .
the reason for these discrepancies is not apparent , but considering the putative epigenetic regulation of 5-lox expression , it is likely that individual life experiences of various colonies of old rodents could result in a colony - specific aging - modified epigenetic regulation of 5-lox expression . regardless of the observed regional differences , all previous studies [ 14 , 17 , 37 ] and our present results are consistent as to the direction ( i.e. , increase ) of aging - associated changes in brain 5-lox expression .
in contrast to the observed effects of aging in brain tissue , we found that in heart tissue aging was associated with a decrease in 5-lox mrna content .
to our knowledge , this is the first time that heart 5-lox expression has been investigated in the context of aging .
previous studies reported that 5-lox content in the heart remains stable during cardiac remodeling   but can be altered by dietary modifications . on the other hand , 5-lox protein content in the aorta
was significantly increased in old ( 24 months ) compared to adult ( 6 months ) rats .          
dna methylation is an epigenetic mechanism involved in the regulation of gene expression including the effects of aging and tissue - specific expression .
we observed that in the heart , which expresses about 50 times greater 5-lox mrna levels than the brain , the dna regions targeted by bstui , hinp1i , and hpaii were less methylated than those in the brain .
typically , methylation of proximal promoters containing a high density of cpg dinucleotides ( cpg islands ) leads to long - term gene silencing .          
. the methylation status of the nonisland cpg loci appears to be equally relevant for gene regulation .
for example , sakamoto et al .   reported that the tissue - dependently and differentially methylated regions were disproportionately distributed in the nonisland cpg loci and that these loci were located not only in 5 regions of genes but also in intronic and nongenic regions .
the mouse 5-lox promoter-5 utr sequence has an equal number ( i.e. , 2 ) of recognition sites for each of the four endonucleases used in our assay .
for example , aging did not affect the methylation status assayed at the hinp1i sites , it only affected the hpaii sites in the hippocampus and produced a similar effect on acii and bstui sites in all tissue samples studied . on the other hand ,
the exon - intron sequence has 4 bstui , 6 hinp1i , 1 hpaii , and 1 acii site .
however , the only effect of aging in this 5-lox dna sequence was observed in brain tissues and only in bstui sites .         
the pattern of dna methylation depends on enzymes such as dnmts , in particular dnmt1 which is involved in the maintenance of dna methylation patterns and dnmt3a which predominantly contributes to de - novo dna methylation .
although the expression of dnmt1 and dnmt3a showed some tissue and regional differences , we did not observe major aging - associated alterations in the mrna levels of these two enzymes , with the only exception that there was   lower dnmt3a mrna content in the heart of old mice .
however , this finding does not exclude the possibility of aging - altered dnmt activity in the brain .
furthermore , multiple enzymatic mechanisms are involved in determining the pattern of dna methylation , including dna - demethylases .
for example , recent findings indicate that neuronal activity is capable of triggering epigenetic dna demethylation , and it remains to be investigated whether this process is affected by aging and whether it could alter 5-lox dna methylation in the absence of significant dnmt changes .        
further research is needed to elucidate if the observed changes in 5-lox dna methylation could play a role in aging - associated modifications of tissue 5-lox mrna levels .
for such a functional role , dna methylation status would have to influence the binding of various methylation - sensitive regulatory factors ( e.g. , transcription activators and repressors ) .
we subjected the 5-lox dna sequences used in our assay ( figure 1 ) to an analysis with the software for searching transcription factor binding sites ( tfbind ) , which uses the weight matrix in transcription factor database transfac r.3.4 .   .
this analysis , along with the survey of available literature , suggests that in the region upstream of the mouse 5-lox atg start codon , the likely candidates are the following transcription factors : ahr / arnt , nmyc , e2f , ap4 , ap2 , usf , and sp1 . in the coding region , putative regulatory factors include ahr / arnt , usf , elk1 , hsf1 , hsf2 , arp1 , rfx1 , gata1 , ap4 , e2f , and arp1 .
nevertheless , one must take into account that the tissue samples we used in this study contained a mixture of cell types and that the link between 5-lox mrna and 5-lox dna methylation may depend on the cell type .
inherent in studies with tissue samples as opposed to research in cell cultures is that 5-lox dna extracted from tissue samples derives from both 5-lox mrna - expressing and mrna - nonexpressing cells .
this technical limitation negatively impacts the ability to directly relate mrna content and dna methylation status .
similarly , cell type may be decisive in determining the type of 5-lox metabolites produced ( e.g. , leukotrienes versus lipoxins ) .         although leukotriene synthesis in the cns and in the heart can occur via a transcellular biosynthetic pathway that is believed not to require 5-lox expression
[ 12 , 13 ] , aging - increased leukotriene synthesis in the hippocampus   and aorta   appears to be associated with increased 5-lox expression .
it was proposed that in the cns , metabolites derived from 5-lox activity could influence neural plasticity , such as the expression of hippocampal long - term depression .
furthermore , it has been established that the biological effects of leukotrienes in the cns   and heart   can be counteracted by noninflammatory 5-lox metabolites lipoxins .
no data are available on the effects of aging on cns and heart production of lipoxins .
this information appears necessary for a full understanding of the biological implications of aging - altered expression of 5-lox in the brain and heart .          in conclusion
, we found that in addition to altering 5-lox mrna content in heart and brain tissues , aging increased 5-lox dna methylation , and also that this effect was dna - site- and tissue - specific .
it has to be stressed that our results were obtained in tissue samples containing a mixture of various cell types and that further insight into functional implications of dna methylation for 5-lox mrna expression would require cell - type directed studies . for example , one possibility is to focus on neuronal dna in brain tissue samples by applying methods for neuronal nuclei isolation .
 the expression of 5-lipoxygenase ( 5-lox ) is affected by aging and regulated by epigenetic mechanisms including dna methylation .   
 we used methylation - sensitive restriction endonucleases ( acii , bstui , hpaii , and hinp1i ) to assess 5-lox dna methylation in brain and heart tissue samples from young ( 2 months ) and old ( 22 months ) mice . 
 we also measured mrna content for 5-lox and the dna methyltransferases dnmt1 and dnmt3a . 
 in young mice , the 5-lox mrna content was significantly greater in the heart compared to the brain ; 5-lox dna methylation was lower , except in the acii assay in which it was higher in the heart . 
 aging decreased 5-lox mrna content in the heart and increased it in the brain . 
 aging also increased 5-lox dna methylation and this effect was site- ( i.e. , enzyme ) and tissue - specific . generally , 
 dnmt1 and dnmt3a mrna content was lower in the brain regions compared to the heart ; the only effect of aging was observed in the mrna content of dnmt3a , which was decreased in the heart of old mice . 
 these results indicate a complex tissue - specific and aging - dependent interplay between the dna methylation system and 5-lox mrna content . 
 interpretation of this data must take into account that the tissue samples contained a mixture of various cell types . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion