reagents . rmpi-1640 ( with glutamax ) was purchased from life technologies ( merelbeke , belgium ) . bsa , h89 , 8-(4-chloro - phenyl - thio)-camp ( cptcamp ) and 8-(4-chloro - phenyl - thio)-2-o - methyl camp ( me - cptcamp ) were purchased from sigma - aldrich ( st . louis , m. ) sb203580 was from biomol ( plymouth , p. ) . except for cptcamp and me - cptcamp , which were dissolved in lps - free water ( baxter , lessines , belgium ) , all inhibitors were dissolved in dmso . the rabbit antibodies against pp38 , erk1/2 , p - jnk , jnk , p - creb and creb , and the mouse monoclonal against p - erk1/2 were obtained from cell signaling ( beverly , mass . ) . p - creb antibody detects creb only when phosphorylated at ser133 and this antibody also detects the phosphorylated form of creb - related protein atf-1 . rabbit antibodies against p38 were from santa cruz biotechnology ( santa cruz , calif . ) . peroxidase - conjugated donkey anti - rabbit and sheep antimouse iggs were obtained from amersham pharmacia biotech ( roosendaal , the netherlands ) . jurkat cells were obtained from the european collection of cell cultures ( salisbury , uk ) . first , pbmcs were isolated by centrifugation on ficoll - isopaque ( pharmacia & upjohn , uppsala , sweden ) density gradients ( 1.077 g / ml ) at 1000 g for 20 min . purity of the t lymphocyte fraction was always over 90% as assessed by flow cytometric analysis . t lymphocytes were resuspended in rpmi-1640 supplemented with 1% bsa , at a concentration of 510/ml . to measure the effects of camp analogues on prl mrna expression , cells were cultured for 6 h in a humidified 5% co2 atmosphere at 37 c . to address camp - induced protein phosphorylation , cells were cultured for 20 min in 2-ml reaction tubes ( eppendorf , hamburg , germany ) in a water bath at 37 c . briefly , for prl mrna detection , a real - time cdna amplification was performed using the applied biosystems ( nieuwerkerk a / d ijssel , the netherlands ) assay - on - demand for human prolactin ( hs 00168730_m1 ) and a five - point standard curve ( 0.02200 ng total rna from jurkat cells ) . this assay uses a specific taqman mgb probe with an fam reporter dye at the 5 end and a nonfluorescent quencher at the 3 end . fluorescence was monitored using the taqman 7700 sequence detector ( applied biosystems ) . for detection of epac1 and epac2 transcripts , cdna samples were subjected to 35 rounds of pcr cycling ( 94 c for 1 min , 50c for 2 min , 72 c for 45 s ) using the following primer sets : for epac1 : cttcctccagaaactctcag ( sense ) and tcagctcatgcgcttcctg ( antisense ) ; for epac2 : ctcattgaacctcacgttcc ( sense ) and agtcatctccttcatgcagg ( antisense ) . blots were stripped by washing in ddh20 for 10 min , followed by a 5-min incubation in 0.2 m naoh and another washing in ddh20 . statistics . statistical differences between groups were determined by anova , followed by tukey s post - test . data represented are means  sd of quadruplicate incubations of cells from one donor . the presented experiments are representative of three ( figs . 1 , 2 , 3 and 5 ) or eight ( fig 1the effect of camp on prl expression in primary t lymphocytes requires de novo mrna synthesis . ( a ) t lymphocytes were preincubated for 1 h with vehicle or the transcriptional inhibitor actinomycin d ( 5 g / ml ) and next stimulated with vehicle or cptcamp ( 250 m ) for 6 h. prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . # significantly different from control without actinomycin d ( p < 0.01 ) . ( b ) decay of prl mrna levels as measured by real - time pcr . t lymphocytes were preincubated for 1 h with actinomycin d ( 5 g / ml ) and next stimulated with vehicle or cptcamp ( 250 m ) for 0 , 4 , 6 and 18 h. prl mrna levels are represented as percentage of control ( actinomycin d 5 g / ml , no cptcamp at time 0 ) . 2effect of inhibition of pka on camp - induced prl mrna expression and creb phosphorylation in human t lymphocytes . ( a ) t lymphocytes were pretreated for 2 h with vehicle , h89 ( 10 m ) or sb203580 ( 50 m ) , before stimulation with 250 m cptcamp for 6 h. prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . control conditions are always significantly different from corresponding cptcamp - stimulated conditions ( p<0.01 ) . ( b ) t lymphocytes were preincubated for 2 h with increasing doses of h89 , before stimulation with cptcamp ( 250 m ) for 20 min . 3effect of camp on mapk activation in human t lymphocytes and role of pka in this effect . ( a ) p - erk , erk , p - jnk and jnk in t lymphocytes stimulated with 250 m of cptcamp . ( b ) p - p38 and total p38 in t lymphocytes stimulated with increasing doses ( 25 , 250 and 500 m ) of cptcamp . ( c ) p - p38 and total p38 in t lymphocytes preincubated for 2 h with increasing doses of h89 , before stimulation with cptcamp ( 250 mm ) . the results in a - c are obtained from three different donors . the results in b detection of epac1 mrna by rt - pcr in human pbmc , t cell and non - t cell samples . lanes 1 , 4 , 7 : pbmc ; lanes 2 , 5 , 8 : t cells ; lanes 3 , 6 , 9 : non - t cells.fig . 5role of epac in the effects of camp on prl expression in human t lymphocytes . ( a ) t cells were stimulated for 20 min with increasing doses ( 25 , 250 and 500 m ) of me - cptcamp . ( b ) t lymphocytes were stimulated for 6 h with increasing doses of cptcamp or mecptcamp . prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . ( c ) p - p38 and total p38 as assessed by western blotting in jurkat cells stimulated for 20 min with vehicle or 250 m of cptcamp . the results in a and b are obtained from two different donors . the results in a the effect of camp on prl expression in primary t lymphocytes requires de novo mrna synthesis . ( a ) t lymphocytes were preincubated for 1 h with vehicle or the transcriptional inhibitor actinomycin d ( 5 g / ml ) and next stimulated with vehicle or cptcamp ( 250 m ) for 6 h. prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . # significantly different from control without actinomycin d ( p < 0.01 ) . ( b ) decay of prl mrna levels as measured by real - time pcr . t lymphocytes were preincubated for 1 h with actinomycin d ( 5 g / ml ) and next stimulated with vehicle or cptcamp ( 250 m ) for 0 , 4 , 6 and 18 h. prl mrna levels are represented as percentage of control ( actinomycin d 5 g / ml , no cptcamp at time 0 ) . effect of inhibition of pka on camp - induced prl mrna expression and creb phosphorylation in human t lymphocytes . ( a ) t lymphocytes were pretreated for 2 h with vehicle , h89 ( 10 m ) or sb203580 ( 50 m ) , before stimulation with 250 m cptcamp for 6 h. prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . control conditions are always significantly different from corresponding cptcamp - stimulated conditions ( p<0.01 ) . ( b ) t lymphocytes were preincubated for 2 h with increasing doses of h89 , before stimulation with cptcamp ( 250 m ) for 20 min . effect of camp on mapk activation in human t lymphocytes and role of pka in this effect . ( a ) p - erk , erk , p - jnk and jnk in t lymphocytes stimulated with 250 m of cptcamp . ( b ) p - p38 and total p38 in t lymphocytes stimulated with increasing doses ( 25 , 250 and 500 m ) of cptcamp . ( c ) p - p38 and total p38 in t lymphocytes preincubated for 2 h with increasing doses of h89 , before stimulation with cptcamp ( 250 mm ) . the results in a - c are obtained from three different donors . the results in b detection of epac1 mrna by rt - pcr in human pbmc , t cell and non - t cell samples . lanes 1 , 4 , 7 : pbmc ; lanes 2 , 5 , 8 : t cells ; lanes 3 , 6 , 9 : non - t cells . role of epac in the effects of camp on prl expression in human t lymphocytes . ( a ) t cells were stimulated for 20 min with increasing doses ( 25 , 250 and 500 m ) of me - cptcamp . ( b ) t lymphocytes were stimulated for 6 h with increasing doses of cptcamp or mecptcamp . prl mrna levels were quantified by real - time pcr and normalized to gapdh mrna expression . ( c ) p - p38 and total p38 as assessed by western blotting in jurkat cells stimulated for 20 min with vehicle or 250 m of cptcamp . the results in a and b are obtained from two different donors . the results in a we previously showed that camp induces prl mrna expression in freshly isolated human pbmcs and in the human t leukemic cell line jurkat . to assess whether camp induces prl expression in primary t lymphocytes , we investigated the effect of camp on prl mrna levels in purified primary human t lymphocytes . we found that the long - acting camp analogue cptcamp stimulated prl mrna levels in primary t lymphocytes by 12.9  11.2-fold . whereas prl mrna levels were positively affected by camp in all investigated donors ( n=12 ) , the size of the response to camp was very much donor dependent , with a 34.7-fold induction in the best responder versus only a 3.6-fold induction in the weakest responder . as shown in figure 1a , preincubation of primary t lymphocytes with the transcriptional inhibitor actinomycin d completely blocked the effect of cptcamp on prl mrna expression . in addition , cptcamp did not affect the rate of decay of the prl message in t cells treated with actinomycin d ( fig . 1b ) , indicating that camp enhances prl expression in primary t lymphocytes by stimulating transcription . camp - induced prl expression is partially mediated via pka in primary human t lymphocytes . we therefore investigated the effect of pka inhibition , using h89 , on camp - induced prl expression in primary t lymphocytes . figure 2a shows that , at maximal subcytotoxic doses ( 10 m ) , h89 only partially blocked cptcamp - induced prl expression , whereas it completely abolished cptcamp - induced phosphorylation of the principal pka target , creb ( fig . role of mapks in camp - induced prl expression . in leukocytes and other cell types , camp has been shown to mediate some of its effects via activation or inhibition of mapk pathways . therefore , since inhibition of pka did not completely block camp - induced expression , we addressed the effect of cptcamp on activation of p38 , erk and jnk . we found that erk and jnk phosphorylation were undetectable both in unstimulated and cptcamp - stimulated t lymphocytes ( fig . 3a ) , but could be induced by 12-o - tetradecanoyl - phorbol-13-acetate ( tpa ) and anisomycin respectively ( data not shown ) . however , cptcamp dose dependently stimulated p38 phosphorylation ( fig . 3b ) . to address the role of pka in the effect of camp on p38 activation , we investigated the effect of h89 on the phosphorylation of p38 . as shown in figure 3c , h89 did not affect cptcamp - induced p38 phosphorylation , indicating cptcamp phosphorylates p38 in a pka - independent manner . the role of the p38 mapk in the effect of cptcamp on prl expression in t lymphocytes was indicated by the finding that sb203580 , a specific p38 inhibitor , partially abolished the effect of cptcamp on prl expression ( fig . whereas pka is the best - known mediator of camp effects , recently , the activation of alternative , pka - independent , signaling routes through epac by camp have been described . we therefore addressed the expression of the novel camp receptors epac1 and epac2 by rt - pcr in primary t lymphocytes of eight donors . epac1 transcripts were amplified from cdna samples obtained from pbmcs and t cells ( as well as from the residual non - t cell fraction ) from all donors ( n=8 ) ( fig . using a methylated cptcamp analogue , which is unable to activate pka , but instead specifically activates epac , we investigated the role of epac in regulating prl expression in t cells . as shown in figure 5a , me - cptcamp stimulated p38 activity in t lymphocytes , whereas it did not affect creb phosphorylation ( data not shown ) . however , unlike cptcamp , which dose dependently stimulated prl mrna levels in human t lymphocytes , me - cptcamp had no effect on prl expression ( fig . we previously demonstrated that the stimulatory effect of cptcamp on prl expression in the human t leukemic cell line jurkat is mediated by pka only . indeed , as depicted in figure 5c , in these cells , cptcamp did not induce p38 phosphorylation . we and others previously showed that camp and factors signaling via camp , such as pge2 , stimulate prl expression through the extrapituitary promoter in human pbmcs and in the human t leukemic cell line jurkat [ 2628 ] . here we show that camp is also a potent inducer of prl transcription in primary t lymphocytes . although camp increased prl expression in all 12 donors tested , there was a large variation in the strength of the response . since in vitro stimulation of prl expression by phytohemagglutinin has been shown to relate to a single nucleotide polymorphisms at 1149 of the lymphoid promoter , the possibility exists that this polymorphism causes a variation in the strength of the camp effect on prl expression . pge2 and other factors that signal through camp , such as catecholamines , usually inhibit th1 responses [ 49 , 50 ] , whereas prl predominantly stimulates expression of th1 cytokines [ 10 , 1215 , 51 ] . since endogenous prl has especially been shown to have immunomodulatory effects in animals subjected to stress [ 52 , 53 ] , one can speculate that induction of prl expression in the immune system serves as a feedback mechanism to restrict the effect of stress hormones on the immune system . this hypothesis could be tested by assessing the effects of catecholamines in the absence and in the presence of inhibiting antibodies against prl . indeed , autocrine or paracrine effects of small amounts of prl secreted by pbmcs have been shown in several studies [ 3 , 54 ] . according to the classical paradigm , however , some of the effects of camp on t lymphocytes have been shown to be pka independent . for example , in a murine th2 cell line   and in human pbmcs , the effects of camp on il-5 expression were pka independent . here we show that in human t lymphocytes , camp stimulates prl gene expression via activation of both pka - dependent and pka - independent signaling pathways . the pka inhibitor h89 completely blocked phosphorylation of creb at ser133 , yet failed to completely inhibit camp - induced prl gene transcription , suggesting that other kinases and transcription factors , beside pka and creb are involved in the effect of camp on prl expression . our observation that camp induced p38 activation , whereas a specific p38 inhibitor , sb203580 , partially inhibited camp - induced - prl expression in t lymphocytes , indicates p38 is one of the kinases that are activated by camp , in addition to pka . for example , camp induces p38 activation in chinese hamster ovary cells , in murine cardiomyocytes , macrophages   and th2 cells , in rat granulosa cells   and in human sk - n - mc neuroblastoma cells . interestingly , whereas in most of these studies the effect of camp on p38 activation was pka dependent , this was not the case in the murine th2 cells . our results indicate that in normal human t lymphocytes , p38 is also activated independently of pka . whereas the signaling intermediate(s ) activated by camp and responsible for the observed p38 activation remain to be identified , the findings that the epac activator me - cptcamp also stimulates p38 phosphorylation , and that primary t lymphocytes express epac1 transcripts , suggest a possible candidate could be the recently identified small guanine nucleotide exchange factor epac1 . epac1 transcripts have only been detected in b lymphocytes   and alveolar macrophages   but not in other leukocyte subpopulations . whereas tiwari et al .   were unable to detect epac1 or epac2 by rt - pcr in human t lymphocytes , we did detect messages for the epac1 gene by rt - pcr in human t cells . since , to date , no specific inhibitors for epac activity are available , proving that epac is indeed responsible for the effect of camp on p38 activation is very difficult . in addition , it has been suggested that in human t cells the camp - induced , but pka - independent , pathway leading to inhibition of il-5 does not involve epac , indicating another , unidentified , signaling protein , which is activated by me - cptcamp as well as by cptcamp , could be responsible for the observed p38 activation . our observation that camp is able to stimulate some prl expression in the presence of h89 , which completely blocks creb phosphorylation , indicates that a pka - independent pathway is also involved in prl expression , and that this pathway is operative in the absence of pka activity . indeed , inhibition of p38 also partially blocked induction of prl expression . however , activation by me - cptcamp of p38 without pka activation was not sufficient to enhance prl expression . these observations suggest that camp induces a third , unidentified , route that stimulates prl expression in human t lymphocytes . the p38 route probably acts in synergy with this unidentified route to enhance prl transcription . in leukemic jurkat t lymphocytes , full induction of prl transcription has previously been demonstrated to require costimulatory signals , which can come from either the pkc - activating phorbolester tpa or the ca ionophore ionomycin [ 26 , 27 ] , in addition to the camp stimulus . these synergistic effects were not observed in normal human pbmcs , where camp potently induced prl expression by itself . we speculate that in human t lymphocytes , camp does not require a costimulus to induce prl expression , because it induces at least one pka - independent pathway , leading to p38 activation , in addition to the well - known pka pathway . interestingly , the p38 pathway was indeed not activated by camp in jurkat cells ( fig . 5c ) but was also activated in the eol-1 cell line . in these cells , as in t lymphocytes , camp by itself is a potent inducer of prl expression . whereas pituitary prl overproduction can be effectively treated using dopamine agonists , leukocyte prl expression is directed by an alternative promoter and is independent from the factors regulating pituitary prl expression . t lymphocyte - derived prl has been implicated in the pathophysiology of auto - immune disease and further research on the signaling routes leading to prl expression in t lymphocytes may be of clinical importance . this paper and earlier publications from us and other groups indicate that camp is an important regulator of leukocyte prl expression . whereas our findings suggest that the pka pathway is important in this process , at least one additional signal transmitted via p38 is required for full prl induction in t lymphocytes . importantly , we have detected the novel camp receptor epac1 in primary t lymphocytes and hypothesize that epac1 could be the signaling intermediate linking camp to p38 .
abstract.beside its pivotal role in reproduction , the pituitary hormone prolactin ( prl ) has been attributed an immunomodulatory function . here 
 we report that camp is an important stimulator of prl transcription in primary human t lymphocytes . 
 inhibition of both protein kinase a ( pka ) and p38 mapk partially abrogated camp - induced prl expression . 
 in addition , camp - induced phosphorylation of p38 was shown to occur independently of pka and could be mimicked by a methylated camp analogue which specifically activates the recently discovered camp receptor epac ( exchange protein directly activated by camp ) . 
 our findings suggest that camp induces prl expression in t lymphocytes via cooperation of at least two different signaling pathways : a pka - dependent pathway leading to the phosphorylation of camp response element - binding protein , and a pka - independent pathway leading to p38 phosphorylation .