the transcriptional factor nuclear factor kappa b ( nf-b ) is a main regulator of the induction of several genes including inflammatory and immune response genes and the proliferation and death of cancer cells [ 1 - 4 ] . cellular stimulation by pro - inflammatory cytokines , gram - negative bacterial lipopolysaccharide ( lps ) , viral infection , radiation , and hypoxia is induced by ib kinase ( ikk ) complex - mediated ib phosphorylation ( predominant form , ikk ) and ubiquitination , and degradation [ 5 - 10 ] . the mammalian nf-b family consists of 5 subunits , p50-nf-b1 , p52-nf-b2 , c - rel , p65-rela , and relb , which forms homo- or heterodimers of functional complexes . the nf-b dimer is mainly sequestered in the cytoplasm as a complex associated with the ib inhibitor in unstimulated resting cells , or rarely in the nucleus as a transcriptionally inactive complex shuttle . in activated cells , ib - free nf-b translocates into the nucleus where it activates the transcription of several genes , including newly synthesized ib . in addition to translocation of nf-b , the transcriptional activities of nf-b are regulated by posttranslational modifications such as phosphorylation and acetylation of p65-nf-b subunit for full activation [ 12 - 14 ] . the transcriptional activities of p65-nf-b , which are specifically targeted by several kinases , are highly enhanced by phosphorylation of ser , or ser , and ser ( by zetapkc ) in association with the coactivator cbp / p300 . additionally , anrather et al .   reported 3 phosphorylation sites at ser ( induced by lps ) , ser ( by msk1 ) , and ser ( by lps and unknown kinase ) as essential phosphorylation sites for transcriptional activity . phosphorylation of p65-nf-b at ser leads to nuclear translocation or cytoplasmic priming , and the transactivation mechanism is the target of several kinases including the important kinase ikk [ 18 - 20 ] . phosphorylation of ser has been studied with other related phosphorylation sites , including ser and ser , to evaluate transactivation [ 17,21 - 23 ] . identified ser , ser , and ser as potential phospho - acceptor sites within the p65 rel homology domain , and found that both ser and ser can be mediated by ser / thr kinases , but ser ser - specific kinases alter transcriptional nf-b activities when ser is substituted with thr . they suggested that the phosphorylation levels of these potential sites can affect the interaction of nf-b with coactivators , and to acetylation patterns for the full nf-b transcriptional activities . additionally , p65-nf-b phosphorylation levels reflect the differential nf-b transcriptional activity of related gene subsets ; however , phosphorylation is not essential for nf-b dna binding . it was previously shown that the transcriptional coactivators p300 and cbp mainly acetylate p65-nf-b lys , lys , and lys . particularly , p65-nf-b lys acetylation stimulates the full transcriptional activity of p65-nf-b , but is only minimally related to dna binding or ib assembly . 5-amp - activated protein kinase ( ampk ) is a highly conserved serine / threonine protein kinase that regulates energy homeostasis and metabolic stress , and exists in all eukaryotic cells as heterotrimeric complexes comprising catalytic -subunits and regulatory - and -subunits . phosphorylation of the thr residue of the  subunit is important for maximum ampk activity [ 26 - 28 ] . lkb1 stimulates ampk in response to changes in the cellular amp / atp ratio , calmodulin - dependent protein kinase kinase  in response to intracellular ca concentration , and transforming growth factor - beta - activating kinase 1 by immunological cytokines [ 29 - 32 ] . in recent years , numerous studies have reported the effects of ampk activities on inflammatory nf-b activity [ 33 - 48 ] . the major function of ampk in inhibiting inflammation has been demonstrated using a known ampk activator and the pharmacological mimetic 5-aminoimidazole-4-carboxamide ribonucleotide ( aicar ) . other studies showed that aicar inhibits tumor necrosis factor ( tnf)- and interleukin--induced nf-b activities in immune cells [ 49 - 52 ] and inducible nitric oxide synthase and cyclooxygenase ( cox-2 ) expression levels in lpsor cytokine - stimulated myocytes , adipocytes , or macrophages grown in culture . however , the anti - inflammatory effects of aicar were also found to be ampk - independent or nonspecific activators of ampk in several studies . in addition to aicar , there are several ampk activators ; metformin , used to treat type 2 diabetes ; berberine , a natural product used in traditional chinese medicine ; and a-769662 , derived from a high - throughput screen for ampk activators . aspirin and salicylate also inhibit the inflammatory nf-b pathway , and it has been proposed that this results from inhibition of the upstream kinase ikk- . however , they suggested that inhibition of the nf-b pathway is mediated by ampk activation , rather than by direct inhibition of ikk-. overall , all ampk activators described above have been reported to inhibit inflammatory responses in various model systems . we recently investigated the effects of lps and the ampk activator aicar on cox-2 induction , two specific p65-nf-b phospho - activities ( ser and ser ) , and the acetylation activity of p65-nf-b lys . particularly , we proposed that the expression levels of p65-nf-b ser phosphorylation and p65-nf-b lys acetylation were inversed , suggesting potential inhibitory activity of p65-nf-b ser phosphorylation . both lpsand hypoxia - induced nf-b activities in a human bladder cancer line t24 were alleviated by pretreatment with aicar . additionally , ampk sirna - mediated suppression enhanced nf-b - mediated cox-2 induction by lps or hypoxia . particularly , we suggested that direct interactions and colocalization occur between p - ampk ( at phospho - activation site thr ) ( p - ampk ) and ib-free nf-b , especially in nucleus . lps - induced full transcriptional activity of nf-b , as indicated by a critical acetylation level ( ac - k310 p65-nf-b ) , was decreased by aicar pretreatment , whereas the phosphorylation level at p65-nf-b ser was increased . we also found that ampk phosphorylation as well as the ability of aicar to enhance phosphorylation of ampk was decreased only at the early time ( ~1 hour ) after lps stimulation . this effect of lps stimulation on p - ampk levels was abolished or showed a greater increase at the later time ( after 16 hours ) . demonstrated that the transient suppression of ampk phosphorylation diminished the ability of aicar to increase ampk phosphorylation in lps - stimulated cells . .   suggested that hmgb1 released form injured or necrotic cells was involved in decreasing lps - treated ampk phospho - activity based on their results showing an inverse relationship between accumulated hmgb1 in cytoplasm and ampk phosphorylation levels . furthermore , we examined n - acetyl cystein ( nac ) pretreatment under the above experimental conditions to immediately inhibit reactive oxygen species ( ros ) release at the early time , which resulted in increased p - ampk levels and decreased cox-2 induction . thus , our finding may also be explained by the influence of early released ros on ampk phospho - activity in lps - stimulated bladder cancer cells . based on our findings , aicar pretreatment partially decreased both lps- and hypoxia - treated cox-2 induction . additionally , lps - induced nf-b p65 ser phospho - activity was decreased by aicar pretreatment , but highly increased by ampk - sirna , suggesting an ampk - dependent mechanism . consistent with this result , recent studies reported the ability of aicar to suppress nf-b activation in response to lps or pro - inflammatory cytokines through an ampk - dependent or -independent mechanism [ 52,59 - 61 ] . it is generally known that the tlr4/nf-b signaling pathway is activated under hypoxic conditions , increasing the gene expression of downstream inflammatory mediators [ 7,8,57,62 - 67 ] . additionally , hypoxia induces ampk activation in cancer cells as a survival mechanism by atp - depletion ( laderoute et al . , 2006 ; miller et al . , 2008 in contrast , the phospho - activities of the serial enzymes p - lkb1 , p - ampk , and p - acc in our study were time - dependently diminished under hypoxia conditions and hif1 expression was increased ( unpublished ) . in addition , p - ampk levels after lps , aicar , or nac treatment and even in untreated cells were decreased under hypoxia conditions , and hypoxia - induced cox-2 expression was synergistically enhanced by additional treatment with lps and blocked or decreased by aicar pretreatment ; these results are similar to those of other recent reports . we recently investigated the effects of lps and the ampk activator aicar on cox-2 induction , two specific p65-nf-b phospho - activities ( ser and ser ) , and the acetylation activity of p65-nf-b lys . particularly , we proposed that the expression levels of p65-nf-b ser phosphorylation and p65-nf-b lys acetylation were inversed , suggesting potential inhibitory activity of p65-nf-b ser phosphorylation . both lpsand hypoxia - induced nf-b activities in a human bladder cancer line t24 were alleviated by pretreatment with aicar . additionally , ampk sirna - mediated suppression enhanced nf-b - mediated cox-2 induction by lps or hypoxia . particularly , we suggested that direct interactions and colocalization occur between p - ampk ( at phospho - activation site thr ) ( p - ampk ) and ib-free nf-b , especially in nucleus . lps - induced full transcriptional activity of nf-b , as indicated by a critical acetylation level ( ac - k310 p65-nf-b ) , was decreased by aicar pretreatment , whereas the phosphorylation level at p65-nf-b ser was increased . we also found that ampk phosphorylation as well as the ability of aicar to enhance phosphorylation of ampk was decreased only at the early time ( ~1 hour ) after lps stimulation . this effect of lps stimulation on p - ampk levels was abolished or showed a greater increase at the later time ( after 16 hours ) . demonstrated that the transient suppression of ampk phosphorylation diminished the ability of aicar to increase ampk phosphorylation in lps - stimulated cells . suggested that hmgb1 released form injured or necrotic cells was involved in decreasing lps - treated ampk phospho - activity based on their results showing an inverse relationship between accumulated hmgb1 in cytoplasm and ampk phosphorylation levels . furthermore , we examined n - acetyl cystein ( nac ) pretreatment under the above experimental conditions to immediately inhibit reactive oxygen species ( ros ) release at the early time , which resulted in increased p - ampk levels and decreased cox-2 induction . thus , our finding may also be explained by the influence of early released ros on ampk phospho - activity in lps - stimulated bladder cancer cells . based on our findings , additionally , lps - induced nf-b p65 ser phospho - activity was decreased by aicar pretreatment , but highly increased by ampk - sirna , suggesting an ampk - dependent mechanism . consistent with this result , recent studies reported the ability of aicar to suppress nf-b activation in response to lps or pro - inflammatory cytokines through an ampk - dependent or -independent mechanism [ 52,59 - 61 ] . it is generally known that the tlr4/nf-b signaling pathway is activated under hypoxic conditions , increasing the gene expression of downstream inflammatory mediators [ 7,8,57,62 - 67 ] . additionally , hypoxia induces ampk activation in cancer cells as a survival mechanism by atp - depletion ( laderoute et al . , 2006 ; miller et al . , 2008 ; kim et al . , 2012 ) . in contrast , the phospho - activities of the serial enzymes p - lkb1 , p - ampk , and p - acc in our study were time - dependently diminished under hypoxia conditions and hif1 expression was increased ( unpublished ) . in addition , p - ampk levels after lps , aicar , or nac treatment and even in untreated cells were decreased under hypoxia conditions , and hypoxia - induced cox-2 expression was synergistically enhanced by additional treatment with lps and blocked or decreased by aicar pretreatment ; these results are similar to those of other recent reports . in this short review , we highlighted the regulatory interactions of ampk activity on nf-b induction , particularly in post - translational phosphorylation and acetylation of nf-b under inflammatory conditions or in hypoxia environments , providing examples in the human bladder cancer cell line t24 .
generally , both lipopolysaccharide ( lps)- and hypoxia - induced nuclear factor kappa b ( nf-b ) effects are alleviated through differential posttranslational modification of nf-b phosphorylation after pretreatment with 5-amp - activated protein kinase ( ampk ) activators such as 5-aminoimidazole-4-carboxamide ribonucleotide ( aicar ) or the hypoglycemic agent metformin . 
 we found that aicar or metformin acts as a regulator of lps / nf-b - or hypoxia / nf-b - mediated cyclooxygenase induction by an ampk - dependent mechanism with interactions between p65-nf-b phosphorylation and acetylation , including in a human bladder cancer cell line ( t24 ) . in summary 
 , we highlighted the regulatory interactions of ampk activity on nf-b induction , particularly in posttranslational phosphorylation and acetylation of nf-b under inflammatory conditions or hypoxia environment .