cell culture  hasmcs ( promocell ) were cultured in smooth muscle cell growth medium 2 supplemented with 5% fetal bovine serum , 0.5 ng / ml recombinant human epidermal growth factor , 2 ng / ml recombinant human basic fibroblast growth factor , 5 mg / ml recombinant human insulin , and 0.62 ng / ml phenol red ( promocell ) . the cells were maintained at 37 c and 5% co2 , and cells in passages 210 were used . isolation of genomic clones for human 1 and 1 sgc subunits  bacterial artificial chromosome clones containing 1 or 1 sgc subunits were purchased from bacpac resources ( children 's hospital oakland research institute ) . the full promoters of human 1 and 1 sgc were isolated from the bacterial artificial chromosome clones by pcr using accuprime taq high fidelity dna polymerase , using primers based on the 5-flanking regions of 1 ( ay034777 ) and 1 ( ay034778 ) sgc genes including the first exon . construction of luciferase deletion plasmids  different 5-flanking regions of the 1 and 1 sgc genes upstream of the transcription start site ( + 1 ) and extending to the noncoding part of the first exon ( + 9 ) were cloned into nhei - xhoi restriction sites of the pgl3-basic luciferase reporter vector ( promega ) . insertions in the pgl3 reporter vector expressing firefly luciferase were identified by restriction digest , and the integrity of the constructs was confirmed by direct sequencing . primer sequences used to generate the full promoter , and deletion constructs are shown in supplemental fig . the a0.3- and b0.5-kb fragments , respectively , were subjected to site - directed mutagenesis by using the quikchange ii site - directed kit ( stratagene ) and high fidelity pfu turbo dna polymerase ( stratagene ) . this kit was also used to repair errors during the construction of luciferase deletion constructs . transient transfections3,500 hasmcs / well and 15,000 cos-7 cells / well were seeded in 96-well clear plates 1 day before transfection . the cells were transfected with 50 ng of plasmid dna / well using fugene 6 reagent ( roche applied science ) with a 3:1 ratio ( l reagent:g dna ) . pgl3 control vector ( promega ) was used as a transfection control . in each case , the cells were co - transfected with a prl - tk vector expressing renilla luciferase ( promega ) at a 10:1 ratio ( test vector : prl - tk vector ) . twenty - four hours later , where indicated cells were treated with either ( a )  pro - inflammatory mixture  ( 100 mg / ml lps ( salmonella typhimurium ; sigma ) , 200 units / ml ifn- , 400 units / ml interleukin-1 , and 2000 units / ml tumor necrosis factor- ( all preprotech ) in cell culture medium ) or ( b ) the no donor deta - nonoate ( deta - no ; 0200 m ) . luciferase assays  forty - eight hours after transfection , the cells were washed in phosphate - buffered saline and lysed for 30 min at room temperature using the passive lysis buffer ( promega ) . luciferase activity was determined using the dual luciferase reporter assay system ( promega ) on the wallac victor 2 multilabel counter ( perkinelmer life sciences ) . firefly luciferase activity was normalized to renilla luciferase derived from prl - tk vector , and the data are expressed as relative luciferase light units . electrophoretic mobility shift assays ( emsa)hasmc plated in 6-cm tissue culture dishes were stimulated for 2 h with the pro - inflammatory mixture or deta - no ( 100 or 200 m ) , nuclear extracts were prepared , and samples were subjected to emsa , as we have previously described ( 35 ) . protein concentrations were determined by bca protein assay ( pierce , distributed by perbio sciences ltd . , the proteins were transferred to a nitrocellulose membrane ( amersham biosciences ) using the mini trans - blot electrophoresis transfer cell ( bio - rad ) . the membranes were stained with ponceau s stain to confirm equal transfer and incubated in 5% milk in wash buffer ( phosphate - buffered saline , 0.1% tween 20 ) overnight at 4 c with gentle shaking . the membranes were then incubated with primary antibody ( anti - sgc 1 ) or anti - glyceraldehyde-3-phosphate dehydrogenase ( abcam , coventry , uk ) diluted 1:2000 ( 1 ) or 1:1000 ( glyceraldehyde-3-phosphate dehydrogenase ) in 5% milk in wash buffer for 3 h at room temperature with constant agitation followed by five washes ( 5 min / wash ) . the membranes were then incubated with shaking for 1 h at room temperature with horseradish peroxidase - conjugated goat anti - rabbit igg ( dako , cambridgeshire , uk ) diluted 1:1000 in 5% milk in wash buffer . the membranes were washed as described above , and the proteins were visualized using enhanced chemiluminescence ( amersham biosciences ) . the bands were quantified by densitometry using alphaimager ( alpha innotech , san leandro , ca ) . cyclic gmp measurements  hasmc ( 10 cells ) were plated in 10-cm dishes and 24 h later exposed to the pro - inflammatory mixture for a further 24 h. at this time , the cells were harvested by trypsinization and centrifuged at 200  g for 10 min . the cell pellet was resuspended in assay buffer ( 2.36 mg/100 ml tris - hcl , 760 mg/100 ml nacl , 22.2 mg/100 ml kcl , 29.6 mg/100 ml mgso4 , 16.6 mg/100 ml na2hpo4 , 0.01 mg/100 ml cacl2 , ph 7.4 ) and treated with 1 mm 3-isobutyl-1-methylxanthine ( sigma ) . the cells were then exposed to either 1 or 10 m spermine - nonoate ( axxora , nottingham , uk ) for 5 min (  basal  samples did not receive spermine - nonoate ) , and the reaction was terminated by heating at 90 c for 10 min . the cyclic gmp content was then measured using a commercially available assay ( amersham biosciences ) . blast was used to identify and retrieve orthologous 1 sgc promoters from mouse , rat , and human genomes from project ensembl at the wellcome trust sanger institute . the 1 sgc upstream sequence was analyzed for the presence of repeat regions and transposable elements , using repeat masker version open-3.1.5 . confirmation of pol ii promoter was conducted with promoter scan ii and novel neural network promoter prediction . identification of cpg islands was performed using cpgprod ( cpg island promoter detection ) software . in silico analysis for transcription factor - binding sites a student 's t test was used to determine differences between data groups , where p < 0.05 was considered significant . the transcriptional start sites were determined by pair - wise blasting of 1 and 1 sgc 5-flanking regions with the complete mrna sequences ( nm_000856 and nm_000857 , respectively ) . scanning both 1 and 1 sgc sequences with the novel neural network algorithms for improved eukaryotic promoter site recognition did not reveal any tata box in close proximity to the transcription start site of both 1 and 1 sgc promoters . analysis of promoter core elements ( 37 ) showed the presence of consensus inr ( initiator ) and dpe ( downstream core promoter ) elements but not tfiib recognition ( bre ) or the newly discovered motif 10 ( mte ) elements in the 1 sgc promoter ( supplemental fig . s3 ) . gc and ccaat - bf boxes were detected in both proximal promoters . furthermore , using repeat masker , we were able to identify three repeats in the 1 sgc promoter but only one in the 1 promoter region ( table 1 ) . finally , cpgprod software did not reveal the presence of cpg islands in either the 1 or 1 sgc promoters . of elementsbeginendlengthpercentage of sequencebphuman 1 sgc promoter repeats according to repeatmaskera  ltr ( malrs )   1   -3038   -2853   185   6.1   dna element ( mer1_type )   1   -2188   -2031   158   5.2   total interspersed repeats   343   11.2   low complexity   1   -80   -42   39   1.3 human 1 sgc promoter repeats according to repeatmaskerb  line1   1   -2429   -1671   759   22.7   total interspersed repeats   759   22.7 atotal length , 3048 bp ; gc level , 36.5% ; based masked , 382 bp ( 12.5%).btotal length , 3344 bp ; gc level , 40.0% ; based masked , 759 bp ( 22.7% ) . the positions are according to the transcription start site ( + 1 ) total length , 3048 bp ; gc level , 36.5% ; based masked , 382 bp ( 12.5% ) . total length , 3344 bp ; gc level , 40.0% ; based masked , 759 bp ( 22.7% ) . transiently transfected luciferase reporter constructs containing 0.33.0 kb of human 1 or 0.33.2 kb of human 1 sgc upstream sequence were analyzed for promoter activity in hasmcs . the a0.3-kb fragment showed the highest level of activity among the 1 sgc promoter constructs ( fig . 1 ) . reduced activity in the remaining ( longer ) constructs suggested the presence of repressor element(s ) further upstream . the b0.5- and b0.8-kb fragments showed the highest level of activity among the 1 sgc promoter constructs ( fig . 1 ) . the b1.4-kb fragment showed a decreased promoter activity , suggesting the presence of a repressor element(s ) between 830 and 1392 bp . extension of the promoter fragment up to 3.2 kb in length did not significantly alter the transcriptional activity of the b1.4-kb construct , suggesting the absence of net activation or repression in that region . however , the 126-bp deletion in b0.4 kb ( to generate construct b0.3-kb ) caused greater than 50% decrease in activity , suggesting that a key , putative enhancer(s ) is located between b0.3 and b0.4-kb . in accord , the a0.3- and b0.5-kb constructs possessed the critical elements necessary for maximal promoter activity of the 1 and 1 sgc promoters , respectively , and were further analyzed . figure 1.promoter activity of human 1 ( a ) and 1 sgc ( b ) deletion constructs in hasmcs . the cells were transfected with luciferase reporter constructs containing different size promoter fragments ( indicated in bp relative to the transcription start site ( + 1 ) ) . promoter activity of each construct was measured as firefly luciferase activity normalized to renilla luciferase , and the results are expressed as relative luciferase light units ( ru ) . promoter activity of human 1 ( a ) and 1 sgc ( b ) deletion constructs in hasmcs . the cells were transfected with luciferase reporter constructs containing different size promoter fragments ( indicated in bp relative to the transcription start site ( + 1 ) ) . promoter activity of each construct was measured as firefly luciferase activity normalized to renilla luciferase , and the results are expressed as relative luciferase light units ( ru ) . figure 2.analysis of putative tfs at the proximal promoter sequence of the human sgc gene under basal conditions in hasmc ( a and c ) and cos-7 ( b and d ) cells . a and b , effects of core deletions of c - myb , gaga , nfat , and ccaat - bf sites on promoter activity of the a0.3-kb construct . c and d , effects of core deletions of nf-b(p50)- , pu1- , sp1- , and nfy - binding sites on promoter activity of the b0.5-kb construct . nfy-1 and nfy-2 indicate the upstream and downstream ccaat - bf sites with nfy-1&2 representing the double deletion . analysis of putative tfs at the proximal promoter sequence of the human sgc gene under basal conditions in hasmc ( a and c ) and cos-7 ( b and d ) cells . a and b , effects of core deletions of c - myb , gaga , nfat , and ccaat - bf sites on promoter activity of the a0.3-kb construct . c and d , effects of core deletions of nf-b(p50)- , pu1- , sp1- , and nfy - binding sites on promoter activity of the b0.5-kb construct . nfy-1 and nfy-2 indicate the upstream and downstream ccaat - bf sites with nfy-1&2 representing the double deletion . interestingly , the a0.3-kb sequence exhibited only 44% homology with the mouse and rat sequence homologues and the b0.5-kb fragment possessed a 57% homology when compared with the rat sequence . as a consequence , there is likely to be disparate regulation of sgc expression ( i.e. promoter activity ) between species , highlighting the importance of characterizing the activity of the human promoter in human cells . identification of critical transcription binding sites  analysis of putative transcription factor - binding sites in the fragments containing the maximal promoter activity for the human 1 and 1 sgc genes was performed using matinspector software ( solution parameters : core similarity , 1.0 ; matrix similarity optimized ) ( supplemental figs . s4a and 5a ) and confirmed by transcription element search software . in both a0.3-kb and b0.5-kb constructs , putative tf - binding sites for several transcription factors located at the optimal regulatory distance from the transcription start site were investigated . to functionally determine the importance of these tfs for 1 and 1 sgc promoter activity , the cores of each tf - binding site were deleted individually in both the a0.3-kb and b0.5-kb basal constructs via site - directed mutagenesis . these deletions did not introduce any new binding sites as confirmed by sequence analysis with matinspector software . transcriptional activity of these mutant constructs was assessed in hasmcs and cos-7 cells ; tf - binding sites that had a significant effect on sgc promoter activity in hasmcs are shown in supplemental figs . a0.3-kb fragments harboring deletions for ccaat - bf- , c - myb- , gaga- , and nfat - binding sites showed significant increases in promoter activity compared with a0.3-kb control in hasmcs ( fig . 2a ) , suggesting that binding of these tfs to their recognition sites has repressor effects on 1 sgc promoter activity in these cells . the effect of c - myb was abolished in cos-7 cells , suggesting a hasmc - specific effect ( fig . moreover , the effects of gaga and nfat were also reduced in cos-7 cells ( fig . 2b ) , despite still having significant repressor activity . however , ccaat - bf had a similar effect in cos-7 cells , suggesting that this tf may belong to the basal transcription machinery for 1 sgc gene in many cell types . b0.5-kb fragments harboring a deletion for an nf-b(p50)-binding site showed almost 2-fold increase in promoter activity compared with b0.5 kb control in hasmcs ( fig . 2d ) , suggesting that nf-b(p50 ) plays a key repressor role in the basal expression of 1 sgc , independent of cell type . in contrast , a b0.5-kb fragment harboring deletions for both nfy - binding sites showed a small reduction in b0.5-kb promoter activity in hasmcs , suggesting a slight activator effect of this tf ( fig . 2c ) . 2d ) and in a be2 human neuroblastoma cell line ( 34 ) , suggesting that a cell - specific effect of nfy is not so apparent in hasmc . pu1 and sp1 tfs showed activator and repressor effects , respectively , in cos-7 cells but not in hasmcs , again suggesting cell - specific regulation of sgc promoter activity . figure 3.temporal effects of a pro - inflammatory mixture on a0.3-kb ( a ) and b0.5-kb ( b ) promoter activity in hasmcs . c , expression of sgc 1 protein determined by western blot in hasmc under basal and pro - inflammatory conditions ( 048 h of incubation with mixture ) . d , basal and no - stimulated increases in cgmp in hasmc in the absence and presence of pro - inflammatory mixture ( 24 h ; n = 40 ) . * , p < 0.05 versus the corresponding response in the absence of pro - inflammatory mixture . temporal effects of a pro - inflammatory mixture on a0.3-kb ( a ) and b0.5-kb ( b ) promoter activity in hasmcs . c , expression of sgc 1 protein determined by western blot in hasmc under basal and pro - inflammatory conditions ( 048 h of incubation with mixture ) . d , basal and no - stimulated increases in cgmp in hasmc in the absence and presence of pro - inflammatory mixture ( 24 h ; n = 40 ) . * , p < 0.05 versus the corresponding response in the absence of pro - inflammatory mixture .   figure 4.electrophoretic mobility shift assay ( a and b ) with densitometric quantification ( c and d ) of hasmc nuclear extracts under basal and pro - inflammatory conditions ( 2h of incubation with the mixture ) was performed using labeled primers carrying the nf-b or ccaat - bf binding sites or the mutated version ( nf-bor ccaat - bf ) . for competition experiments , a 100 excess of unlabeled double - stranded oligonucleotide ( cold probe ) was added to the binding reaction . a supershift at the nf-b site was demonstrable using a p50 , but not p65 , antibody ( e ) . electrophoretic mobility shift assay ( a and b ) with densitometric quantification ( c and d ) of hasmc nuclear extracts under basal and pro - inflammatory conditions ( 2h of incubation with the mixture ) was performed using labeled primers carrying the nf-b or ccaat - bf binding sites or the mutated version ( nf-bor ccaat - bf ) . for competition experiments , a 100 excess of unlabeled double - stranded oligonucleotide ( cold probe ) was added to the binding reaction . a supershift at the nf-b site was demonstrable using a p50 , but not p65 , antibody ( e ) . ( n  3 ) . * , p < 0.05 . effect of pro - inflammatory conditions on sgc promoter activity in hasmcs  to examine whether vascular inflammation modulates sgc expression in hasmcs , human 1 and 1 sgc promoter activity was examined in the presence of a pro - inflammatory mixture using the a0.3-kb ( fig . pro - inflammatory conditions significantly reduced both the 1 and 1 sgc promoter activity at 24 h. moreover , western blot analysis revealed a clear , time - dependent decrease in sgc 1 expression following exposure of the hasmc to the pro - inflammatory mixture ( fig . 3c ) that was mirrored by a significant reduction in both the basal and no - stimulated sgc activity ( fig . 2 , ccaat - bf and nf-b exerted the most pronounced effect on 1 and 1 sgc promoter activity , respectively , among the tfs studied . to determine whether these transcription factors bind to the target dna sequences on the promoter , nuclear extracts from hasmcs were analyzed by emsa . in terms of the 1 sgc promoter , three complexes were detected using the oligonucleotide probe containing 93 to 66 bp region ( ccaat - bf oligonucleotide ) upstream of the transcriptional start site ( fig . the specificity of these complexes was demonstrated by a reduction of the intensity of all three bands in the presence of an excess of unlabeled probe . when the nuclear extract derived from hasmcs treated with the pro - inflammatory mixture was analyzed , an increase in the intensity of two of these complexes was observed ( fig . 4c ) , suggesting that vascular inflammation increased the binding of ccaat - bf to the proximal 1 sgc promoter . the deletion of a single c ( g ) of the core of the ccaat - bf site ( position 82 ; supplemental fig . s4 ) abolished the formation of two of the three complexes , highlighting the importance of that single base for the binding of the ccaat - bf to the target promoter . figure 5.concentration- ( a and b ) and time- ( c and d ) dependent effects of the no donor deta - no ( 0200 m and 048-h incubation ) on a0.3 ( a and c ) and b0.5 ( b and d ) promoter activity in hasmcs . ( n  3 ) . * , p < 0.05 versus response in the absence of deta - no . concentration- ( a and b ) and time- ( c and d ) dependent effects of the no donor deta - no ( 0200 m and 048-h incubation ) on a0.3 ( a and c ) and b0.5 ( b and d ) promoter activity in hasmcs . promoter activity was measured as described in the legend to fig . ( n  3 ) . * , p < 0.05 versus response in the absence of deta - no . for the 1 sgc promoter , interactions within the nf-b(p50)-binding region were also assessed using a nf-b oligonucleotide encompassing 138 to 109 bp region of this promoter ( supplemental fig . these complexes were no longer formed in the presence of an excess of unlabeled probe , confirming specificity ( fig . interestingly , a supershift of the complexes was only observed when we used an anti - p50 antibody but not anti - p65 ( fig . 4e ) , suggesting that the smaller nf-b subunit ( perhaps as a p50 homodimer ) is pivotal in regulating sgc 1 expression ; this finding dovetails well with the observed increase in sgc 1 promoter activity in the construct with deleted p50 consensus site(s ) and with previous reports detailing a predominantly repressor activity of p50 homodimers ( 38 , 39 ) . when the nuclear extract derived from hasmcs treated with the pro - inflammatory mixture was analyzed , an increase in the intensity of the complex bands was observed ( fig . 4d ) , suggesting that vascular inflammation increased the binding of the nf-b(p50 ) factor to the proximal 1 sgc promoter . the deletion of the 4-bp core ( gggg ) for the nf-b factor - binding site disrupted the formation of two complexes and reduced the formation of the third one , indicating that the gggg motif is necessary for its dna interaction . furthermore , an important role for nf-b in regulating the expression of the 1 subunit was supported by a parallel reduction in enzyme protein expression and activity ( fig . 3 , c and d ) . effect of no on sgc promoter activity in hasmcs  to assess the effect of the sgc ligand no on expression of the enzyme in hasmcs ( to establish the existence of a feedback loop in terms of enzyme expression , which is well established to occur biochemically ( 30 , 40 ) ) , human 1 and 1 sgc promoter activity was examined in the presence of the no donor deta - nonoate ( releases no spontaneously in aqueous solution ( 41 ) ) . pilot studies revealed that for the 1 subunit , the promoter activity was inhibited to the greatest extent in the a0.3 construct , suggesting that key no - dependent repressors are located in this region of the promoter . for 1 sgc , maximal inhibition was observed in the b0.5 construct , suggesting that this area of the promoter contains the critical no - responsive repressor element(s ) . 5 shows that no exerted a concentration - dependent inhibition of both 1 and 1 sgc promoter activity . this effect was maximal at 200 m deta - no , which results in an approximate ambient no concentration of 200 nm ( 42 ) . moreover , the inhibition of promoter activity by no followed a time course very similar to that observed under pro - inflammatory conditions with maximal effect observed at 2448 h ( fig . 5 ) . to determine whether the repressor activity of no was cgmp - dependent or independent , further experiments were conducted using the a0.3 and b0.5 constructs as models of promoter function . here , the no - dependent inhibition of both the 1 and 1 sgc promoter was not reversed in the presence of the sgc blocker odq ( 5 m ) nor mimicked by the cgmp analogue 8-br - cgmp ( 300 m ; fig . thus , it appears that the inhibitory effect of no on sgc promoter activity and expression is mediated directly , rather than via activation of the enzyme ( and cgmp production ) . in an attempt to identify putative transcription factors that might mediate the repressor effects of no , a0.3 , and b0.5 constructs with targeted mutations in specific tf - binding sites were analyzed for their responsiveness to no . interestingly , the activity of the a0.3 fragment with a deleted ccaat - bf site was no longer sensitive to inhibition by deta - no ( fig . 6c ) , intimating that this response element is involved in mediating the negative feedback effect of no on sgc expression . in contrast , deletion of the same site in the b0.5 construct did not alter promoter activity in the presence of no . figure 6.effect of the sgc inhibitor odq ( 5 m ) and cgmp analogue 8br - cgmp ( 300 m ) and core deletions of ccaat - bf and nfy on no - mediated ( deta - no , 200 m ; 24 h of incubation ) inhibition of a0.3 ( a and c ) and b0.5 ( b and d ) promoter activity in hasmc . promoter activity was measured as described in the legend to fig . ( n  3 ) . * , p < 0.05 versus respective control . # , p < 0.05 versus a0.3 + deta . in c and d , promoter activity in the absence of deta is normalized to 100% . effect of the sgc inhibitor odq ( 5 m ) and cgmp analogue 8br - cgmp ( 300 m ) and core deletions of ccaat - bf and nfy on no - mediated ( deta - no , 200 m ; 24 h of incubation ) inhibition of a0.3 ( a and c ) and b0.5 ( b and d ) promoter activity in hasmc . ( n  3 ) . * , p < 0.05 versus respective control . # , p < 0.05 versus a0.3 + deta . in c and d , promoter activity in the absence of deta is normalized to 100% . figure 7.electrophoretic mobility shift assay ( a ) with densitometric quantification ( b ) of hasmc nuclear extracts under basal conditions and in the presence of 100 m deta - no ( 2-h incubation ) was performed using labeled primers carrying the ccaat - bf site or its mutated version ( ccaat - bf ) . for competition experiments , a 100 excess of unlabeled doubled - stranded oligonucleotide ( cold probe ) was added to the binding reaction . electrophoretic mobility shift assay ( a ) with densitometric quantification ( b ) of hasmc nuclear extracts under basal conditions and in the presence of 100 m deta - no ( 2-h incubation ) was performed using labeled primers carrying the ccaat - bf site or its mutated version ( ccaat - bf ) . for competition experiments , a 100 excess of unlabeled doubled - stranded oligonucleotide ( cold probe ) was added to the binding reaction . finally , we confirmed the importance of no in inhibiting sgc promoter activity via the ccaat - bf site , by emsa . in accord with the luciferase assay data , deta - no ( 100 m ) caused an increase in the intensity of the three ccaat - bf complexes observed that was essentially absent when using probes with a dysfunctional ccaat - bf site ( fig . however , despite attempting to produce a supershift in the ccaat - bf complexes observed with specific antibodies to two putative ccaat - bfs ( e.g. cbf - b , maz ) , we were unable to observe such a change ( data not shown ) , suggesting that additional ccaat - bfs are likely involved . activation of sgc is essential for no - mediated regulation of vascular smooth muscle reactivity ( 5 ) and proliferation ( 6 ) , microvascular permeability ( 7 , 8) , and the reactivity / adherence of platelets ( 9 ) and leukocytes ( 8 , 11 ) . as such , under physiological circumstances activation of sgc is fundamental to cardiovascular homeostasis and maintains an important cytoprotective / anti - thrombotic influence . moreover , because inappropriate sgc activity may be responsible for several features of cardiovascular pathologies including blood pressure dysregulation ( i.e. smooth muscle reactivity ) , capillary edema , and cellular recruitment , dysfunction of sgc is likely to have at least an equivalent impact on ( patho)physiology as inappropriate no production ; changes in the expressional regulation ( and activity ) of this enzyme are therefore likely to exert considerable influence on the progression of disease . despite this obvious importance , there exists scant information concerning the physiological regulation and pathological alterations of sgc expression . to address this deficit , we have characterized the human 1 and 1 sgc promoter regions in human aortic smooth muscle cells . analysis of the human 1 and 1 sgc gene promoters revealed some intriguing aspects of transcriptional regulation of these genes and differences in the promoter consensus core elements . both sgc genes have a tata - less promoter and show ccaat - bf sites in direct ( 1 sgc ) or reverse ( 1 sgc ) orientation 80 bp upstream from the putative transcription start site , in agreement with the canonical position for eukaryotic promoters ( 43 ) . classically , the following three - way combinations are highly favorable in human promoter regions to facilitate gene transcription : bre - inr - mte and/or tata - inr - mte ( 37 ) ; yet , neither combination was detected in either the 1 or 1 sgc promoter . however , because the 1 sgc promoter exhibits two potential initiation modules , an alternative transcription start site 51 bp downstream given by the inr - dpe module ( and characteristic of tata - less promoters ) ( 44 ) can not be ruled out . functional analysis of constructs containing different size fragments of the 1 and 1 sgc 5-flanking regions showed a slightly different profile of activity between both subunit promoters in hasmcs . despite 1 sgc showing the highest promoter activity in the a0.3-kb construct , suggesting repressor elements localized further upstream , the situation was different for 1 sgc . here , the b0.5-kb construct showed the highest promoter activity , and the presence of several enhancers and repressors functioning at a distance from this regulatory region was detected . the fact that b0.5-kb activity was comparable with b0.8 kb activity in hasmcs but has been shown to be over 3-fold lower than b0.8 kb in be2 cells ( 34 ) , suggests a cell - specific regulation of the crucial promoter region necessary to achieve the maximum 1 sgc promoter activity in hasmcs . this cell - specific effect is also highlighted by the fact that b0.4-kb activity is almost the same as b0.5 kb and 2-fold higher than b0.3 kb in hasmcs , intimating strong activator elements located in the 253 to 354 region , not observed in be2 cells ( 34 ) . thus , a0.3-kb and b0.5-kb constructs demonstrated the highest promoter activity in hasmcs , indicating that the regulatory promoter of sgc has all the necessary elements for maximum promoter activity in the 300500-bp region upstream of the transcription start site . we also focused on the identification of principal tfs responsible for the transcriptional regulation of 1 and 1 sgc in hasmcs . a multiplicity of putative transcription factor - binding sites predicted by matinspector software are clustered in both a0.3-kb ( 1 sgc ) and b0.5-kb ( 1 sgc ) promoter fragments , all of which have been shown to play an important role in the transcription of a wide variety of eukaryotic genes . gata ( gata - binding factor , typical of the cardiovascular system ) , gfi1 ( growth factor independence i ; typical of the hematopoietic system ) , nfy ( ubiquitous ) , and pbx ( homeodomain factor ; typical of the hematopoietic system ) have consensus sequences in both promoters , and some of these factors may have a regulatory role in the co - expression of both 1 and 1 sgc genes ; this is particularly pertinent for the gfi1 ( both 1 and 1 sgc ) and pbx ( 1 sgc ) sites , which are positioned between the ccaat - bf site and the transcription start site and therefore are likely to play key roles in expressional regulation of sgc in hematopoiesis . indeed , the 1 and 1 sgc promoter regions contain no less than 20 consensus sequences for tfs reported to play a role in regulating hematopoiesis , supporting a key role for sgc in this process ( 34 ) . functional analysis with a luciferase 1 sgc reporter construct containing a c - myb , gaga , nfat , or ccaat - bf core deletion in the a0.3-kb promoter fragment demonstrated that the integrity of these sites is very important for transcription repression in hasmcs . the c - myb and to a lesser extent the gaga and nfat repressor effects were not observed in cos-7 cells , intimating a vascular smooth muscle cell - specific effect . in fact , c - myb plays a crucial role in arterial smooth muscle cell proliferation ( associated with g1/s cell cycle transition ) , as occurs in many vasculopathies ( 45 ) and nfat is associated with cardiac morphogenesis , vasculogenesis , and vascular smooth muscle hypertrophy ( 46 ) ; the physiological importance of no - sgc signaling in curbing these proliferative processes fits with the ability of such tfs to suppress sgc promoter activity and thereby promote pathogenesis . however , it is noteworthy that when two other nfat sites located at 174 and 161 in 1 sgc were mutated , they did not significantly alter 1 sgc promoter activity . functional analysis with a luciferase 1 sgc reporter construct containing a nfy core deletion in the b0.5-kb promoter fragment revealed that this site is critical for transcription activation in hasmcs , although this effect was less evident than in cos-7 cells ( this study ) and be2 cells ( 34 ) . similar analysis also showed that the integrity of an nf-b(p50 ) site is very important for transcription repression control , in both hasmcs and cos-7 cells , suggesting that this tf is globally important in constitutive gene expression . to corroborate the importance of these tfs in regulation of sgc promoter activity , we conducted emsas to confirm the interaction of ccaat - bf and nf-b with the sgc promoter region . it is clear from these data that both ccaat - bf and nf-b(p50 ) bind strongly to the 1 and 1 sgc promoter constructs , respectively , and that the effects of these tfs on sgc promoter activity are specific . a ccaat - bf site has been found to be prevalent in the promoters of cell cycle - regulated eukaryotic genes ( 47 ) and is known to be essential for cell cycle - dependent activation and repression of several mammalian genes ( 4850 ) . ( 34 ) , a second upstream site was also shown to have an important role , highlighting the role nfy plays in 1 sgc transcription . moreover , the presence of the two closely situated nfy sites in the immediate promoter is in agreement with the idea that expression of 1 sgc gene could be regulated during the cell cycle ( 34 ) . many cardiovascular diseases , particularly atherosclerosis , are now recognized to constitute a chronic inflammation of the blood vessel wall . because no - sgc - cgmp signaling exerts an important vasoprotective , anti - atherogenic effect , elucidation of the expressional regulation of sgc in a pro - inflammatory environment is likely to provide further insight into the pathogenesis of vascular disease . to mimic such a pathological scenario in the vasculature , we examined the effect of pro - inflammatory conditions on sgc promoter activity in hasmcs . a0.3-kb and under such conditions , sgc a0.3-kb and b0.5-kb promoter activity at 24 h was significantly reduced , demonstrating a coordinated regulation of both 1 and 1 sgc gene expression . the fact that the strongest repressors for 1 ad 1 sgc ( ccaat - bf and nf-b , respectively ) exhibit enhanced dna binding under pro - inflammatory conditions might explain , at least in part , the reduction in 1 and 1 sgc promoter activity observed under this  pathogenic  environment . moreover , analysis of protein expression confirmed that the decreased promoter activity in the presence of pro - inflammatory mixture was mirrored by reductions in both sgc 1 protein expression and activity , providing a key link between nf-b and regulation of sgc bioactivity . we also examined the effect of no per se to mediate a feedback loop and prevent sgc promoter activity / expression . using the no donor deta - no , we demonstrated that no exerts a concentration - dependent reduction in 1 and 1 sgc promoter activity . in these studies , the inhibitory effect of no peaked at 200 m deta - no , which results in an ambient no concentration of 200 nm ( 42 ) , more akin to those associated with inducible no synthase activity and pathological conditions ( rather than generated by constitutive no synthase ) moreover , the effects of no in this respect appear to be cgmp - independent , implying direct effects on tf activity ( as has been shown for nf-b ( 51 ) and hif-1 ( 52 ) ) . these studies also revealed that a ccaat - bf site is pivotal to the inhibitory effects of no , at least in terms of the 1 subunit , because deletion of this site leads to insensitivity to no . because several tfs bind this site , it is difficult to pinpoint which might be responsible ; despite attempts to use emsa - based supershifts to derive information concerning the identity of this tf , we were unable to elucidate the mechanism further ( although we did obtain negative results for cbf - b and maz , suggesting that it is not either of these factors ) . the inflammation- and no - based reduction in sgc promoter activity and protein expression might be likely to represent a common negative feedback regulation of sgc expression in cardiovascular disorders associated with high output no production , such as septic shock . here , it would be appropriate for sgc expression to be down - regulated to offset the excessive no levels and thereby reverse the associated , life - threatening hypotension . these data also suggest that the reduction in sgc mrna levels reported in vascular smooth muscle cells in response to lps / interleukin-1 ( 27 ) , tumor necrosis factor-/lps ( 53 ) , and no donors ( 54 , 55 ) is likely to be due , at least in part , to a direct down - regulation in sgc promoter activity via nf-b(p50)- and ccaat - bf - dependent pathways . in sum , these data provide a systematic , comparative analysis of human sgc promoter regulation in hasmcs and has identified potentially important factors regulating human sgc expression within a cell system relevant to cardiovascular physiology and ( patho)physiology . these observations suggest that expressional regulation of human sgc 1 and 1 subunit expression , both coordinated and individually , is likely to play an important role in the ( patho)physiological regulation of enzyme activity . as such , these findings represent a significant advance in the understanding of expressional regulation of the genes encoding sgc subunits and provide insight into potential pathogenic mechanisms that result in aberrant no - sgc signaling .
soluble guanylyl cyclase ( sgc ) is the principal receptor for no and plays a ubiquitous role in regulating cellular function . 
 this is exemplified in the cardiovascular system where sgc governs smooth muscle tone and growth , vascular permeability , leukocyte flux , and platelet aggregation . as a consequence 
 , aberrant no - sgc signaling has been linked to diseases including hypertension , atherosclerosis , and stroke . despite these key ( patho)physiological roles , 
 little is known about the expressional regulation of sgc . to address this deficit , 
 we have characterized the promoter activity of human 1 and 1 sgc genes in a cell type relevant to cardiovascular ( patho)physiology , primary human aortic smooth muscle cells . 
 luciferase reporter constructs revealed that the 0.3- and 0.5-kb regions upstream of the transcription start sites were optimal for 1 and 1 sgc promoter activity , respectively . 
 deletion of consensus sites for c - myb , gaga , nfat , nf-b(p50 ) , and ccaat - binding factor(s ) ( ccaat - bf ) revealed that these are the principal transcription factors regulating basal sgc expression . 
 in addition , under pro - inflammatory conditions , the effects of the strongest 1 and 1 sgc repressors were enhanced , and enzyme expression and activity were reduced ; in particular , nf-b(p50 ) is pivotal in regulating enzyme expression under such conditions . 
 no itself also elicited a cgmp - independent negative feedback effect on sgc promoter activity that is mediated , in part , via ccaat - bf activity . in sum , these data provide a systematic characterization of the promoter activity of human sgc 1 and 1 subunits and identify key transcription factors that govern subunit expression under basal and pro - inflammatory ( i.e. atherogenic ) conditions and in the presence of ligand no .