tissue - type plasminogen activator ( t - pa ) converts plasminogen into plasmin , which in turn dissolves the fibrin structure of thrombi . the fibrinolytic properties of t - pa have been widely studied , but it has become evident that the role for t - pa in the brain extends beyond that of regulating vascular patency . in the brain , t - pa is produced by neurons and glial cells1 and is involved in physiological and pathophysiological processes , including various aspects of synaptic plasticity and neurodegeneration.24 in the context of ischemic stroke , aside from the beneficial effects of t - pa as a thrombolytic , much attention has been drawn to its proposed neurotoxic effects . following excessive neuronal depolarization or glial activation this may result in neurotoxicity and initiation of deleterious proteolytic cascades.1,3 indeed , endogenous t - pa has been shown to potentiate the extent of ischemic injury,5,6 while astrocyte - derived pai-1 can rescue neurons from these effects.7 an understanding of the role and regulation of t - pa and its inhibitors in the cns may provide crucial tools for the development of novel neuroprotective strategies . however , as studies addressing these issues are difficult to perform in humans , a great majority of the studies conducted to date have utilized in vivo and in vitro animal models . in view of species - specific structural differences between the t - pa and pai-1 promoters , the question remains as to how pertinent the results obtained in these animal models can be translated into the human brain . in an attempt to examine this issue , we have compared the effects of physiological and pathophysiological stimuli , as well as activators of protein kinases , on the expression of the t - pa and pai-1 genes in astrocytes of human and rat origin . human astrocytes ( clonetics , walkersville , md ) were cultured in astrocyte growth medium ( agm ) supplemented with 3% fetal bovine serum ( fbs ) as described.8 all experimental series were performed using astrocytes from two to three individuals . primary cortical rat astrocytes were prepared from newborn ( p1p2 ) sprague - dawley rats according to protocols approved by the ethics committee of the university of gothenburg and cultured as described.9 human and rat astrocyte cultures were > 95% glial fibrillary acidic protein ( gfap ) positive . human or rat astrocytes were seeded in 12-well dishes and allowed to reach 100% confluency . cells were incubated in culture medium containing retinoic acid ( ra , 10 m ) , phorbol 12-myristate 13-acetate ( pma , 10 m ) , forskolin ( 2  10 m ) , interleukin-6 ( il-6 , 10 ng / ml ) , tumor necrosis factor- ( tnf- , 5 ng / ml ) , transforming growth factor- ( tgf- , 1 ng / ml ) , or dimethyl sulfoxide ( dmso ) vehicle ( 0.2% ) for the indicated time periods . for oxygen glucose deprivation ( ogd ) , cells were transferred to a hypoxic chamber ( biospherix , redfield , ny ) and maintained under hypoxic conditions ( 1% o2 ) in serum - free and glucose - free dulbecco modified eagle medium ( dmem ) . control cultures were maintained under normal culturing conditions in serum - free dmem containing glucose . three treatment series were performed on two separate occasions ( n = 6 ) . following treatment , cells were lysed with lysis buffer ( buffer rlt ; qiagen , hilden , germany ) , and lysates were stored at 70c . total cellular rna was isolated using an rneasy mini kit ( qiagen ) and mrna was converted to cdna with the geneamp rna pcr kit ( applied biosystems , foster city , ca ) . target mrna was quantified by taqman real - time rt - pcr and normalized relative to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) mrna or 18s , for ogd experiments , according to a standardized protocol.8,10,11 the oligonucleotide primers and taqman probes for quantification of human t - pa , pai-1 , gapdh , and 18s mrna were as follows : t - pa forward primer ( fp ) 5-ggccttgtctcctttctattcg-3 , t - pa reverse primer ( rp ) 5-agcggctggatgggtacag-3 ; t - pa probe ( pr ) 5-(fam)tgacatgagcctccttcagccgct(tamra)-3 , pai-1 ( fp ) 5-ggctgacttcacgagtctttca-3 ; pai-1 ( rp ) 5-ttcactttctgcagcgcct-30 , pai-1 ( pr ) 5-(fam)accaagagcctctccacgtcgcg(tamra)-3 ; gapdh ( fp ) 5-ccacatcgctcagacaccat-3 , gapdh ( rp ) 5-ccaggcgcccaatacg-3 ; gapdh ( pr ) 5-(fam)aaggtgaaggtcggagtcaacggatttg(tamra)-3 ; and applied biosystems assay - on - demand hs99999901_s1 . for amplification of rat t - pa , pai-1 , gapdh , and 18s applied biosystems assay - on - demand rn01482584_m1 , rn01481341_m1 , rn01462662_g1 , and taqman , eukaryotic 18s rrna endogenous control was used . values are presented as mean and standard error of the mean ( sem ) , and a p - value of 0.05 or less was considered statistically significant . differences in the mrna levels between human and rat cultures were evaluated by the unpaired student s t - test ( spss inc . , chicago , il ) . human astrocytes ( clonetics , walkersville , md ) were cultured in astrocyte growth medium ( agm ) supplemented with 3% fetal bovine serum ( fbs ) as described.8 all experimental series were performed using astrocytes from two to three individuals . primary cortical rat astrocytes were prepared from newborn ( p1p2 ) sprague - dawley rats according to protocols approved by the ethics committee of the university of gothenburg and cultured as described.9 human and rat astrocyte cultures were > 95% glial fibrillary acidic protein ( gfap ) positive . human or rat astrocytes were seeded in 12-well dishes and allowed to reach 100% confluency . cells were incubated in culture medium containing retinoic acid ( ra , 10 m ) , phorbol 12-myristate 13-acetate ( pma , 10 m ) , forskolin ( 2  10 m ) , interleukin-6 ( il-6 , 10 ng / ml ) , tumor necrosis factor- ( tnf- , 5 ng / ml ) , transforming growth factor- ( tgf- , 1 ng / ml ) , or dimethyl sulfoxide ( dmso ) vehicle ( 0.2% ) for the indicated time periods . for oxygen glucose deprivation ( ogd ) , cells were transferred to a hypoxic chamber ( biospherix , redfield , ny ) and maintained under hypoxic conditions ( 1% o2 ) in serum - free and glucose - free dulbecco modified eagle medium ( dmem ) . control cultures were maintained under normal culturing conditions in serum - free dmem containing glucose . following treatment , cells were lysed with lysis buffer ( buffer rlt ; qiagen , hilden , germany ) , and lysates were stored at 70c . total cellular rna was isolated using an rneasy mini kit ( qiagen ) and mrna was converted to cdna with the geneamp rna pcr kit ( applied biosystems , foster city , ca ) . target mrna was quantified by taqman real - time rt - pcr and normalized relative to glyceraldehyde 3-phosphate dehydrogenase ( gapdh ) mrna or 18s , for ogd experiments , according to a standardized protocol.8,10,11 the oligonucleotide primers and taqman probes for quantification of human t - pa , pai-1 , gapdh , and 18s mrna were as follows : t - pa forward primer ( fp ) 5-ggccttgtctcctttctattcg-3 , t - pa reverse primer ( rp ) 5-agcggctggatgggtacag-3 ; t - pa probe ( pr ) 5-(fam)tgacatgagcctccttcagccgct(tamra)-3 , pai-1 ( fp ) 5-ggctgacttcacgagtctttca-3 ; pai-1 ( rp ) 5-ttcactttctgcagcgcct-30 , pai-1 ( pr ) 5-(fam)accaagagcctctccacgtcgcg(tamra)-3 ; gapdh ( fp ) 5-ccacatcgctcagacaccat-3 , gapdh ( rp ) 5-ccaggcgcccaatacg-3 ; gapdh ( pr ) 5-(fam)aaggtgaaggtcggagtcaacggatttg(tamra)-3 ; and applied biosystems assay - on - demand hs99999901_s1 . for amplification of rat t - pa , pai-1 , gapdh , and 18s applied biosystems assay - on - demand rn01482584_m1 , rn01481341_m1 , rn01462662_g1 , and taqman , eukaryotic 18s rrna endogenous control was used . values are presented as mean and standard error of the mean ( sem ) , and a p - value of 0.05 or less was considered statistically significant . differences in the mrna levels between human and rat cultures were evaluated by the unpaired student s t - test ( spss inc . , chicago , il ) . with regard to t - pa , the most pronounced difference between human and rat astrocytes was observed in response to treatment with the protein kinase a ( pka ) activator forskolin ( table 1a ) . in human cells , forskolin caused more than a 50% reduction in t - pa expression compared to control - treated cells , whereas t - pa expression was strongly ( > 4-fold ) induced in rat astrocytes . species - specific differences in t - pa response following camp activation have also been described for other cell types.12 we have shown that the protein kinase c ( pkc ) activator pma and ra induce t - pa expression in human astrocytes8 ( data included in table 1a for comparative purposes ) . in rat astrocytes , t - pa expression in response to these stimuli was markedly different as t - pa mrna levels were only weakly increased or even down - regulated following treatment with pma or ra ( table 1a ) . with regard to pai-1 , pkc activation resulted in the most pronounced difference , where prolonged pma treatment ( 20 hours ) induced a 14-fold induction in rat pai-1 expression , while human pai-1 levels were unchanged ( table 1b ) . significant differences between human and rat pai-1 induction were also observed following prolonged ogd ( table 1b ) . an additional series of experiments were performed with cytokines ( human tgf- , tnf- , and il-6 ) , all of which resulted in significant species - specific differences in t - pa and pai-1 , eg , 20 hours treatment with tgf- resulted in a 30% t - pa mrna reduction in human cells and a 4-fold induction in rat cells ( table 1a ) , whereas 20 hours treatment with tnf- reduced human pai-1 expression and enhanced rat pai-1 expression 4-fold ( table 1b ) . our findings are in agreement with studies on other cell types showing that pai-1 regulation in response to cytokines and hypoxia is highly complex , involving different gene regulatory elements and transcription factors depending on cell type and/or species.13 a potential mechanism behind species - specific differences in t - pa and pai-1 gene expression is dna sequence variations . indeed , the rat t - pa promoter contains a perfect camp - responsive element ( cre ) consensus sequence ( tgacgtca ) , whereas the human and mouse counterparts contain a single nucleotide substitution in the core of the motif ( ga , figure 1a ) , potentially affecting both transcriptional responsiveness to camp - elevating agents and cre - binding protein ( creb ) binding affinity.12,14 in line with this , we observed a strong induction of rat t - pa and a reduced expression of human t - pa following treatment with forskolin . as several studies show that this cre - like site is a key dna element mediating human t - pa gene transcription following pkc activation,1417 it is also possible that the sequence variability at this site contributes to the differential levels of t - pa gene activation detected in the human and rat astrocytes . the observed opposite species - specific responses following pka and pkc activation may seem contradictory given that both pka and pkc activators act through the cre , yet pk a treatment induced high expression of rat t - pa and reduced human t - pa whereas pkc treatment produces the opposite effect . however , it should be noted that the one base difference in the core of the human cre - like element changes it to another transcription factor consensus sequence specifically recognized by ap1,18 a trans - acting factor mediating pkc - induced gene expression . in line with this , studies on human endothelial and hela cells show that treatment with pma results in a selective recruitment of ap1 family members fos and jun to the cre - like site.15,16 thus , while the rat cre , as opposed to that of the human cre - like site , provides a high affinity binding site for creb , it is possible that this single nucleotide substitution in the human element renders this site more responsive to pkc - mediated transactivation . if so , this could provide a plausible explanation behind the higher level of t - pa expression observed in human astrocytes compared to that of the rat following treatment with pma . in this respect , studies addressing cell type - specific patterns of t - pa gene regulation following pma treatment show that differences in the binding affinity of individual transcription factors associated with the human cre - like site correlate with the degree of pma - induced t - pa promoter activation.15,17 it is also of interest to note that a suppression of t - pa in ht1080 cells mediated by pma was associated with binding of creb-1 to the cre - like site.17 in addition , functional analyzes of the human t - pa promoter show that the gc - boxes located in the proximal part of the promoter , particularly gc - box iii , are important for pma - induced t - pa transcription,1416 and a possible involvement of the ccaat - binding transcription factor / nuclear factor-1 ( ctf / nf-1 ) site can not be excluded.15 whether the species - specific sequence dissimilarities at these sites affect transcription factor binding affinity and t - pa promoter inducibility remains to be determined . in terms of pai-1 , the strikingly different level of induction observed in human versus rat cells following pma treatment was unexpected . previous studies show that the transcriptional pai-1 response to pma may be mediated via ap1 and sp1 sites in the proximal promoter ( fig . 1b).19,20 given the high degree of sequence conservation in these regions , it seems likely that additional regulatory elements are involved in this response , and this remains to be explored . in this context , it should also be noted that our real - time rt - pcr data do not necessarily reflect changes in gene transcription . thus , it is also possible that additional regulatory control may be exerted post - transcriptionally , for instance at the level of mrna stability , and this may also be subject to species variation . a significant difference in pai-1 expression was also observed following prolonged ogd treatment ( 2.6-fold induction of rat pai-1 and 28% reduction in human pai-1 , table 1b ) . our results are supported by other studies showing species - specific effects in pai-1 gene regulation and induction following hypoxia . while rat pai-1 is induced at very mild levels of hypoxia ( 8% o2),21 severe hypoxia ( < 2% o2 ) was required to induce human pai-1 expression.22 the trans- and cis - acting factors involved in the pai-1 response to hypoxia have been extensively studied . collectively , data suggest a highly complex regulatory pattern , which is dependent on not only species but also cell type , hypoxia level , and context . hypoxia - inducible factor-1 ( hif-1 ) is a central trans - acting factor conveying hypoxia - mediated pai-1 induction , but several other important factors have been identified , such as usf-2 , hif-2 , egr-1 , and net ( reviewed by dimova and kietzmann13 ) . both the human and rat pai-1 promoters contain several potential hypoxia - responsive elements ( hres ) . while the gene regulatory mechanisms conveying the response of the pai-1 gene to hypoxia in astrocytes remain to be established , studies on other human cell types suggest an involvement of several cis - elements , particularly hre2 and two distally located e - boxes ( e4 and e5).22,23 these two e - boxes are not present in the rat promoter ( fig . the two main gene regulatory elements conveying the hypoxia response appear to be two hres : rhre1 and rhre2,21,24 the later of which shares almost perfect sequence homology with the human hre1 ( fig . however , because of the one base substitution in the core of the human motif , the human hre1 lacks binding of hypoxia - inducible factors and is unable to convey hypoxia - induced promoter activation.22 in this context , it should also be noted that hif-1 may be activated by other stimuli , such as hormones and cytokines.25 thus , species - specific differences in the elements binding hif-1 may also affect pai-1 gene responses under normoxic conditions . furthermore , marked quantitative and qualitative differences in the trans - acting factors that mediate the pai-1 response to hypoxia have been described , and shown to be dependent on both cell type and species.2124 it should be added that in addition to the possibility of species - specific changes in post - transcriptional regulation , there may also be differential epigenetic mechanisms controlling the regulation of these genes but this is yet to be explored . in summary , the exact mechanisms behind the observed species - specific differences in the response to the factors assayed in the present report remain to be established , but the clear sequence variation in important transcription factor binding sites ( fig . other mechanisms may include species - specific differences in the levels of transcription factors , co - factors , and second messenger systems involved in signal transduction , as well as epigenetic mechanisms . another consideration relates to species - specific post - transcriptional regulatory events controlling t - pa and pai-1 gene expressions , ie , changes in mrna stability that are well known to be influenced by changes in sequence of the transcript itself , mostly in the 3-utr . while this was not specifically evaluated in this study , the fact that we have identified marked species - specific changes in t - pa and pai-1 gene expression at the mrna level in response to various agonists underlies the difficulties in interpreting results from rodents to humans . there are marked quantitative and qualitative species - specific differences in the responses of the t - pa and pai-1 genes in astrocytes . whether the same is true for other species , including mouse , remains to be established . in view of the present data , although the use of rodent models may provide helpful research tools for scientists seeking mechanisms behind disease and potential targets for future therapies , extrapolations to the in vivo situation in humans need to be made with caution .
in recent years , the role and physiological regulation of the serine protease tissue - type plasminogen activator ( t - pa ) and its inhibitors , including plasminogen activator inhibitor type-1 ( pai-1 ) , in the brain have received much attention . however , as studies focusing these issues are difficult to perform in humans , a great majority of the studies conducted to date have utilized rodent in vivo and/or in vitro models . in view of the species - specific structural differences present in both the t - pa and the pai-1 promoters , we have compared the response of these genes in astrocytes of rat and human origin . 
 we reveal marked quantitative and qualitative species - specific differences in gene induction following treatment with various physiological and pathological stimuli . 
 thus , our findings are of importance for the interpretation of previous and future results related to t - pa and pai-1 expression .