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Regulation of prolactin expression in leukemic cell lines and peripheral blood mononuclear cells
Journal of Neuroimmunology 135 (2003) 107 – 116 www.elsevier.com/locate/jneuroim
Regulation of prolactin expression in leukemic cell lines and peripheral blood mononuclear cells Sarah Gerlo a,*, Wim Vanden Berghe b, Peggy Verdood a, Elizabeth L. Hooghe-Peters a, Ron Kooijman a a
Department of Pharmacology, Free University of Brussels (V.U.B.), Laarbeeklaan 103, B-1090 Brussels, Belgium b Department of Molecular Biology, University of Ghent, Ledeganckstraat 35, 9000 Ghent, Belgium Received 21 August 2002; received in revised form 25 November 2002; accepted 25 November 2002
Abstract To address the role of different intracellular signals in prolactin (PRL) expression in leukocytes, we have investigated the effects of chlorophenylthio-cAMP (cptcAMP), phorbol myristate acetate (PMA) and ionomycin on the activation of the upstream PRL promoter in several leukemic cell lines. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. In freshly isolated peripheral blood mononuclear cells (PBMC), PRL expression could only be stimulated by cptcAMP. The physiological importance of cAMP in the regulation of PRL expression in leukocytes is suggested by the finding that in PBMC, PRL expression is enhanced by prostaglandin-E2 and the h2-adrenergic agonist terbutaline, which both signal through cAMP. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prolactin; Leukocytes; PMA; cAMP; Calcium; Prostaglandins
1. Introduction PRL is a 23-kDa polypeptide hormone that is produced mainly by the anterior lobe of the pituitary. The best known function of pituitary-derived PRL is the initiation and maintenance of lactation. However, PRL is a pleiotropic and versatile hormone that also plays a role in reproduction, angiogenesis and modulation of immune responses. Indeed, PRL receptors are widely distributed in a variety of tissues. Furthermore, several extrapituitary sources of PRL have been described and it is now well established that prolactin is expressed in the decidua, the brain, the mammary gland and in cells of the immune system (Ben Jonathan et al., 1996; Kooijman et al., 2000a). The fact that PRL subserves many different functions can, in part, be explained by tissuespecific regulation of PRL expression. Regulation of PRL expression in the pituitary has been extensively studied, but the regulation of ectopic PRL expression has remained relatively unexplored. The classical modulators of pituitary PRL expression [i.e. dopamine, thyrotropin releasing hormone (TRH) and estrogen] are unable to regulate extrapituitary PRL expression (Gellersen et al., 1989; Handwerger * Corresponding author. Tel.: +32-2-477-4462; fax: +32-2-477-4464. E-mail address: [email protected] (S. Gerlo).
et al., 1991; Bonhoff and Gellersen, 1994). This can be explained by the finding that in humans transcription in extrapituitary tissues is directed by an alternative promoter located 5.8 kb upstream of the pituitary-specific start site (Berwaer et al., 1994; Gellersen et al., 1994). Due to the presence of a 5Vnon-coding exon (exon 1a), the extrapituitary transcript of the PRL gene is 150 nucleotides longer than its pituitary counterpart. The PRL peptide produced by decidua and lymphocytes is, however, indistinguishable from that of pituitary origin. In the immune system, the PRL receptor, which belongs to the cytokine-receptor superfamily, is expressed on T-cells, B-cells, monocytes, and NK-cells (Pellegrini et al., 1992; Matera, 1996). Although PRL does not seem to be required for immune responses under normal circumstances in mice (Horseman et al., 1997), there is nevertheless compelling evidence for modulatory actions of PRL on both humoraland cell-mediated immune responses. For instance, PRL enhances the expression of inducible nitric oxide synthetase, immunoglobulins, and cytokines in human leukocytes (Dogusan et al., 2000; Jacobi et al., 2001; Lahat et al., 1993; Matera et al., 1999; Cesario et al., 1994). The immunomodulatory effects of PRL suggest that it plays a role in the communication between the neuroendocrine system and the immune system. Indeed, pituitary PRL secretion is regulated
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(02)00438-1
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by endotoxin, cytokines and during T-cell-dependent immune responses (Theas et al., 1998, 2001; Perez et al., 1999). Furthermore, local effects of PRL in the immune system are suggested by the expression of PRL in many different types of leukocytes. For instance, PRL mRNA was found to be expressed in thymocytes, T-cells, and B-cells (Pellegrini et al., 1992; Montgomery, 2001). Whereas leukocyte-derived PRL may be involved in normal immune responses, the regulation of PRL expression in leukocytes has hardly been addressed. In peripheral blood mononuclear cells (PBMC), PRL expression is stimulated by phytohaemagglutinin (PHA) and concanavalin A (Con A) (20 –21). Reem et al. (1999) have shown that in the leukemic T-cell line Jurkat, the upstream PRL promoter is activated by cptcAMP, a long-lasting cAMP analogue, and that this activation is synergistically influenced by the phorbolester PMA. A CRE at 25 bp in the upstream PRL promoter is partially responsible for this induction. Additionally, enhanced PRL production in leukocytes has been observed in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and in some patients with acute myeloid leukemia (AML) (Larrea et al., 1997; Gutierrez et al., 1995; Kooijman et al., 2000b). It has furthermore been suggested that PRL produced by leukemic blasts can contribute to enhanced serum PRL concentrations (Hatfill et al., 1990; Ales and Byrd, 2001). These findings indicate that modulation of extrapituitary PRL expression might play a role in the pathophysiology of immune and hematological disorders and prompted the present investigations on the regulation of PRL expression in the immune system. The present study was designed to further investigate the regulation of PRL in different leukemic cells and in PBMC from normal donors. To address intracellular signals leading to promoter activation, we have studied the effects of PMA, cAMP, and the Ca2 + ionophore ionomycin in leukemic cells that were transfected with reporter plasmids carrying the upstream PRL promoter. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. The effects on promoter activation in transfected Jurkat cells reflected regulation of endogenous PRL expression. In the PBMC, only cAMP stimulated PRL gene expression, whereas PMA and ionomycin were inhibitory. A similar response was found in the PRL secreting myeloid cell line Eol-1. We furthermore show that extracellular stimuli, which use cAMP as a second messenger (PGE2 and the h2-agonist terbutaline), enhance PRL expression in PBMC.
antiserum VLS-2 was donated by Y.N. Sinha (San Diego, CA). Human PRL cDNA was provided by J. Martial (Lie`ge, Belgium). The human T-leukemic cell line Jurkat, the human B-lymphoblastoid cell line IM-9, and the myeloid leukemic cell lines Eol-1, K562, and U937 were obtained from the European Collection of Cell Cultures (Salisbury, UK). PGE2, cptcAMP, ionomycin, PGE2, and terbutaline were purchased from Calbiochem (Darmstadt, Germany). DEAE-dextran and PMA were from Sigma (Bornem, Belgium). Recombinant hPRL was produced in Escherichia coli in our lab using a cDNA obtained from J. Martial (Lie`ge, Belgium). GF109203X was purchased from Biomol (Plymouth, PA). 2.2. Subjects Blood donors were between 25 and 55 years of age. Informed consent was obtained from all blood donors and the research protocol has been approved by the local ethical committee. 2.3. Cell preparation and cell culture Human PBMC were purified from heparinized venous blood drawn from healthy donors. PBMC were isolated by centrifugation on Ficoll-Isopaque (Pharmacia & Upjohn, Uppsala, Sweden) density gradients (1.077 g/ml) at 1000 g for 20 min at room temperature and subsequently resuspended in RPMI at a concentration of 107/ml. Cells were cultured in Falcon polystyrene six-well plates (Beckton Dickinson Labware Europe, France). Jurkat, IM-9, and Eol-1 cells were maintained in RPMI supplemented with 10% FCS, K562, and U937 cells in RPMI supplemented with 10% NBCS. For stimulation experiments, cell lines were resuspended in RPMI at a concentration of 2 106/ ml and cultured in Falcon polystyrene 6- or 24-well plates (Beckton Dickinson Labware). All cell cultures were done in a humidified 5% CO2 atmosphere at 37 jC. To assess regulation of prolactin expression, cells were cultured with the appropriate stimuli in RPMI for 18 h. PMA, cptcAMP, and ionomycin were used at concentrations of 10 ng/ml, 250 AM, or 200 ng/ml, respectively. cptcAMP was dissolved in ddH2O, PMA, and ionomycin in dimethylsulfoxide (DMSO). In all conditions, the final DMSO concentration never exceeded 0.02% and was without effect. 2.4. Plasmid constructs
2. Materials and methods 2.1. Reagents RMPI 1640 (with glutamax), newborn calf serum (NBCS), and fetal calf serum (FCS) were purchased from Life Technologies (Merelbeke, Belgium). Anti-hPRL rabbit
The plasmid construct PRL-1842-Luc, containing the region between 1842 and + 35 of the upstream PRL promoter inserted into the pGL3 enhancer reporter plasmid (Promega, Madison, WI), was obtained from A. Stevens and J. Davis (Manchester, UK). Deletion constructs were prepared using fragments of the 1842/ + 35 region that were generated by digestion with restriction enzymes. The
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pGL3 enhancer vector was linearized using SmaI and NcoI and restriction fragments with appropriately modified ends were inserted by semi-blunt ligation. The PRL-3000Luc construct was a gift from B. Gellersen (Hamburg, Germany). 2.5. Transient transfections Jurkat, U937, IM-9, and K562 cells were transfected by electroporation. Cells growing in the log phase were washed and resuspended in RPMI at a concentration of 20 106 cells/ml. Plasmids were added to a concentration of 20 Ag/ml and electroporation was performed using the following settings: 330 V and 1800 AF for Jurkat, 300 V and 1800 AF for U937 and IM-9, and 300 V and 1500 AF for K562 (Electropore 2000, ‘‘home-made’’ device, laboratory of J. Martial, Lie`ge, Belgium). Following electroporation, cells were resuspended in culture medium. After a recovery period of 24 h, cells were washed and resuspended in RPMI at a concentration of 1 106/ml. Subsequently, cells were cultured in 24-well plates (0.5 ml/ well) for 18 h in the presence of the appropriate stimuli. Cell lysates were prepared and assayed for luciferase activity using the Promega Luciferase Assay kit (Promega), following the manufacturer’s instructions. Luminescence was measured using a Biocounter M 1500 luminometer (Celsis, Landgraaf, The Netherlands). Data were calculated as luciferase activity per milligram protein (BioRad protein assay, Bio-Rad, Munich, Germany) and were expressed as fold induction relative to the untreated samples.
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2.8. Western blotting Conditioned media were concentrated 50 times using Ultrafree MC concentrators (Millipore, Bedford, MA), after which protein content was determined using the Bio-Rad protein assay (Bio-Rad). Equal amounts of protein were subjected to polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h in blocking buffer (0.05% Tween20 and 2% non-fat dry milk in PBS), blots were incubated with the rabbit anti-PRL serum VLS-2 (1:4000 in blocking buffer) for 18 h at 4 jC. Subsequently, blots were washed three times for 10 min in PBS/0.5% Tween-20 and incubated with a peroxidase-conjugated goat anti-rabbit IgG antiserum (1:10,000 in blocking buffer). After three more washes, antigen – antibody complexes were detected using Western Lightning detection reagent (Perkin Elmer Life Sciences, Boston, MA) according to the manufacturer’s instructions. 2.9. Statistical analysis Statistical differences between group means were determined by ANOVA with Tukey’s post-test. Differences were considered significant when p < 0.05. Data represented are means F S.D. of at least three independent experiments.
3. Results 3.1. Activation of the upstream PRL promoter by PMA, cptcAMP, and ionomycin
2.6. Reverse transcription-PCR and Southern blotting Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Gibco-BRL). For PCR, we used a sense primer corresponding to a sequence within exon 1a (5V-GAG-ACA-CCA-AGA-AGA-ATC-GGA-3V) and an antisense primer corresponding to a sequence in exon 5 (5V-ATG-ATT-CGG-CAC-TTC-AGG-AGC-3V). Reverse transcription was performed as described earlier (Dogusan et al., 2001). For PCR amplification of PRL cDNA, reaction mixtures were subjected to 35 cycles of PCR (denaturation at 94 jC for 60 s, annealing at 55 jC for 60 s, and extension at 72 jC for 90 s). PCR for GADPH was performed as described earlier (Dogusan et al., 2001). 2.7. PRL assays PRL concentrations in conditioned media were determined using an electrochemiluminescence immunoassay (ECLIA; Elecsys 1010/2010 Systems) from Roche Diagnostics (Manheim, Germany). Monoclonal antibodies used in this sandwich ELISA do not cross-react with hGH, hHCG, hPL, TSH, FSH, or LH. The detection limit of this assay is 0.5 ng/ml.
Promoter activation was assessed using leukemic cell lines that were transiently transfected with a PRL-1842-Luc construct. As depicted in Fig. 1, PMA stimulates promoter activation in the Jurkat T-cell line, the IM-9 B-lymphoblastoid cell line, the K562 erythroblastoid cell line, and the myelomonocytic cell line U937. Since PMA is known to stimulate protein kinase C (PKC) activity, we assessed the effects of PMA in the presence of the PKC inhibitor GF109203X, a bisindolylmaleimide with high specificity for PKC as compared to other protein kinases (Toullec et al., 1991). Stimulation of promoter activity in Jurkat, K562, U937, and IM-9 cells was blocked by this inhibitor, whereas basal promoter activity was not affected (Fig. 1). Deletion of sequences 1842/ 1662 or 1842/ 967 of our promoter construct resulted in the disappearance of PMA effects in K562, U937, and IM-9 cells but not in Jurkat cells (Fig. 2), indicating the presence of a ‘‘PMA responsive element’’ in the distal region between coordinates 1842 and 1662, which is operative in K562, U937, and IM-9 cells and of another ‘‘PMA responsive element’’ between 967 and + 35, which is operative in Jurkat. In Jurkat cells, the cAMP analogue cptcAMP stimulates promoter activation by 2-fold in the absence of other factors,
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Fig. 1. PMA induces activation of PRL-1842-Luc in several leukemic cell lines and this activation is inhibited by the PKC inhibitor GF109203X. Jurkat, IM-9, K562, and U937 cells were transiently transfected with PRL1842-Luc. Cells were preincubated for 1 h with 1 AM (for Jurkat, K562, and U937) or 10 AM (IM-9) GF109203X, before stimulation with 10 ng/ml PMA. After 18 h, cells were harvested. Reporter gene activation is expressed as fold induction relative to unstimulated control values. PMA promoter activation (versus control) and inhibition of PMA activation by GF109203X were significant ( p < 0.05) in all cell lines.
and exerts a strong synergistic effect with PMA (Fig. 3). These results are in accordance with the results of Reem et al. (1999) using Jurkat cells transfected with a 375Luc PRL promoter construct. The synergistic effects of cptcAMP and PMA were still observed in Jurkat cells transfected with 1662, 1519, or 967-Luc constructs (data not shown). cptcAMP also stimulated PRL-1842-Luc in IM-9 cells by 3-fold, but in these cells PMA and cptcAMP have additive rather than synergistic effects. Remarkably, no effects of cptcAMP were observed in K562 and U937 cells (Fig. 3). The Ca2 + ionophore ionomycin alone did not affect basal promoter activity, but interfered with promoter activation by PMA or cptcAMP. In Jurkat cells, ionomycin strongly augmented promoter activation in synergism with cptcAMP. The cptcAMP effect in IM-9 cells was unaffected by ionomycin. Both in Jurkat and
Fig. 2. Effects of PMA on activation of PRL-1842-Luc constructs with 5V deletions. Jurkat, IM-9, K562, and U937 cells were transiently transfected with equimolar amounts of PRL-1842-Luc, PRL-1662-Luc, PRL-1519Luc, and PRL-967-Luc. Reporter gene activation is expressed as fold induction relative to unstimulated control values. The effects of PMA on constructs with 5Vdeletions were significantly ( p < 0.05) different from PMA activation of the PRL-1842-Luc construct in all cell lines.
Fig. 3. Cell-specific activation of the upstream PRL promoter by PMA, cptcAMP, and ionomycin. Jurkat, IM-9, K562, and U937 cells were transiently transfected with PRL-1842-Luc. Reporter gene activation is expressed as fold induction relative to unstimulated control values. (a) Different from unstimulated control with p < 0.001; (b) Different from parallel condition without ionomycin with p < 0.001; (c) Different from parallel condition without ionomycin with p < 0.05.
IM-9 cells, the effect of the combination of PMA, cptcAMP, and ionomycin on promoter activation was smaller than that of PMA and cptcAMP in IM-9, or that of cptcAMP with either PMA or ionomycin in Jurkat cells. Furthermore,
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endogenous PRL. As we have chosen the sense PCR primer in exon 1a, we have only amplified the long, decidual PRL (dPRL) message and not the short, pituitary PRL mRNA. Fig. 4A shows that PRL mRNA levels were moderately stimulated by PMA or cptcAMP. Combinations of cptcAMP with either PMA or ionomycin induced a strong stimulation of mRNA expression, which is in accordance with a strong induction of promoter activation in transfection studies (Fig. 1). Also in accordance with promoter studies is the smaller stimulation of mRNA expression when all three stimuli are added. Next, we studied the effects on PRL secretion by measuring PRL levels in conditioned medium after an 18-h culture period. We found a small stimulation of PRL secretion by either PMA or cptcAMP, and a strong stimulation by cptcAMP in combination with either PMA or ionomycin (Fig. 4B). Since the production of immunoreactive PRL variants by leukocytes has been described in various studies (Sabharwal et al., 1992; Larrea et al.,
Fig. 4. Regulation of PRL expression in Jurkat by PMA, cptcAMP, and ionomycin. (A) Regulation at the mRNA level as assessed by RT-PCR. PCR amplified products were subjected to agarose gel electrophoresis. Densitometric scanning analysis was performed on ethidium bromide stained gels and PRL mRNA quantity was corrected for differences in GADPH mRNA content. The histogram represents the mean values from three independent experiments, whereas the gels are representative of three independent experiments. (B) Regulation of PRL secretion as assessed by ECLIA. (C) Detection of 23 kDa PRL in conditioned media by Western blotting; 23 kDa PRL was not detected when normal rabbit serum was used (not shown). Specificity of the binding of anti-PRL antiserum was confirmed by preincubation of the antiserum with 0.1 Ag of rhPRL for 1 h at 37 jC. The data in B and C represent two different experiments.
ionomycin slightly inhibits PMA promoter activation in K562 and IM-9, but not in Jurkat and U937 cells (Fig. 3). All stimuli were used at concentrations that did not affect cell viability as assessed by trypan blue exclusion. 3.2. Regulation of endogenous PRL in cell lines To address whether the effects of cptcAMP, PMA, and ionomycin on promoter activation are representative for regulation of endogenous PRL, we investigated the effects of these stimulators on PRL mRNA expression and PRL secretion by Jurkat cells, because this cell line expresses
Fig. 5. Regulation of PRL expression in Eol-1 by PMA, cptcAMP, and ionomycin. (A) Regulation of mRNA, assessed and quantified as described in Fig. 4. (B) Regulation of PRL secretion as assessed by ECLIA. (C) Detection of 23 kDa PRL in conditioned media by Western blotting as described in Fig. 4. The data in B and C represent two different experiments.
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1997), we assessed the molecular weight of immunoreactive PRL that was secreted upon stimulation with PMA, cptcAMP, or ionomycin by Western blot analysis. We found that PMA or cptcAMP alone weakly stimulates the expression of 23 kDa PRL, whereas the combinations of cptcAMP with PMA or ionomycin strongly activate 23 kDa PRL and also weakly induce the secretion of 25 kDa PRL (Fig. 4C). Taken together, these results indicate that promoter activation in Jurkat parallels mRNA expression and the secretion of 23 kDa PRL. To further address endogenous regulation of PRL in leukocytes, we investigated regulation of mRNA levels and protein secretion in another leukemic (eosinophilic) cell line, Eol-1, that expresses high levels of PRL (Kooijman et al., 2000b). Activation studies using this cell line revealed that cptcAMP markedly increased PRL mRNA levels, whereas PMA and ionomycin were without effect (Fig. 5A). Addition of PMA slightly reduced the stimulation by cptcAMP, whereas the presence of ionomycin completely abrogated the effect of cptcAMP. Protein studies revealed that PRL secretion generally reflected mRNA levels and was mainly stimulated by cptcAMP alone (Fig. 5B). In addition, Western blot analysis showed that cptcAMP only stimulated the secretion of 23 kDa PRL (Fig. 5C). Thus, in Eol-1 cells, mRNA levels and secretion of 23 kDa PRL were mainly regulated by cAMP. However, promoter activation in Eol-1 cells could not be established, because we have been so far unable to transfect these cells efficiently.
Fig. 7. Regulation of PRL mRNA expression by PGE2 and terbutaline in normal PBMC. Expression of mRNA was measured by RT-PCR, followed by Southern blotting. The results are representative of donors that responded to PGE2 (4 out of 6) and terbutaline (3 out of 4).
whereas PMA always reduced basal mRNA levels. Remarkably, the effect of cptcAMP was completely inhibited by PMA in six out of seven donors, whereas a synergistic effect of cptcAMP and PMA was observed in one donor (Fig. 6C). Notably, the magnitude of the stimulating effects of cptcAMP were variable (Fig. 6A vs. B) and the synergistic effect was observed in the donor with the smallest response to cptcAMP alone (Fig. 6C). The effects of ionomycin were assayed in PBMC from four donors, and it appeared that ionomycin, alone or in combination with either PMA or cptcAMP, inhibited PRL expression (Fig. 6A: 5 –8).
3.3. Regulation of PRL expression in normal human PBMC To assess the regulation of PRL expression in normal leukocytes, we analyzed the effects of cptcAMP, PMA, and ionomycin on freshly isolated PBMC from seven normal donors. We found that cptcAMP increased PRL mRNA expression in PBMC from all seven donors tested (Fig. 6),
Fig. 6. Regulation of PRL mRNA expression in PBMC. Expression of mRNA was measured by RT-PCR, followed by Southern blotting. Characteristic responses to stimulation with PMA (2), cptcAMP (3), and PMA + cptcAMP (4) as compared to the unstimulated control (1) are represented for three donors (A, B, and C). The effect of ionomycin (5), ionomycin + PMA (6), ionomycin + cptcAMP (7), or ionomycin + PMA + cptcAMP (8), was similar in all donors tested and is shown for donor A.
Fig. 8. Regulation of PRL promoter activation and endogenous PRL expression by PGE2 in Jurkat. Jurkat cells were stimulated for 18 h with 1, 10, and 100 nM PGE2. (A) Jurkat cells transiently transfected with PRL1842-Luc. (B) PCR amplified products were subjected to agarose gel electrophoresis and visualised by ethidium bromide staining. (C) PRL secretion as assessed by ECLIA.
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3.4. Stimulation of PRL expression by PGE2 and the 2adrenergic receptor agonist terbutaline The finding that cAMP is a major regulator of PRL mRNA expression in PBMC and some leukemic cell lines prompted us to study the effects of physiological modulators of the immune system which operate through cAMP as a second messenger. PGE2 has been implicated in the regulation of both cellular and humoral immune responses (Goodwin and Ceuppens, 1983) and operates via cAMP and other second messengers. To test the possible effects of h-adrenergic receptors, which contribute to neuromodulation of immune responses (Xu, 2001), we incubated cells with the h2-adrenergic receptor agonist terbutaline. As shown in Fig. 7, 10 –100 AM PGE2 increased PRL mRNA levels in PBMC of four out of six donors, whereas 1 – 10 AM terbutaline did so in three out of four donors. In Jurkat cells, terbutaline had no effect on PRL expression (data not shown), whereas PGE2 increased PRL mRNA levels (Fig. 8B). In Jurkat cells, PGE2 also stimulated promoter activation (Fig. 8A) and induced a concomitant increase in PRL secretion (Fig. 8C).
4. Discussion The regulation of prolactin expression in leukocytes is different from that seen in the pituitary. The basis of this divergent cell-specific transcription appears to be the use of an alternative promoter in extrapituitary tissues such as the decidua and leukocytes (Berwaer et al., 1994; Gellersen et al., 1994). In the present study, we show that a promoter construct, carrying 1842 bp of the upstream PRL promoter, was activated by PMA through activation of PKC in all leukocytic cell lines we investigated. Reem et al. (1999) did not find activation of the upstream PRL promoter with PMA alone in Jurkat, which can be explained by the fact that they used a construct carrying only 375 bp of the upstream promoter. Indeed, more recently, they showed PMA induction of upstream PRL promoter activity in Jurkat using the PRL-1842-Luc construct (Stevens et al., 2001). The observations of Reem et al. (1999) and our finding that PMA responsiveness is retained with a 967-Luc PRL promoter construct, suggest that another PMA responsive element is located between 967 and 375 of the upstream PRL promoter. In K562, U937, and IM-9 cells, the responsive element for the PMA effect is localised between 1842 and 1662 in the upstream PRL promoter. Watanabe et al. (2001) have described an AP-1 site at 1705 in the upstream PRL promoter that appeared to function as an enhancer in decidualised endometrial stromal cells but not in non-decidual cells. The authors did not find activation of this enhancer region when it was transfected into Jurkat cells, nor did they find a footprint using Jurkat cell nuclear extract. Our findings support the idea that the 1705 AP-1 site in the upstream PRL promoter is not functional in Jurkat
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cells. However, it could play a role in other leukemic cells such as K562, U937, and IM-9 cells. In Jurkat cells, a third responsive element in the region 375/ + 35 acts synergistically with a CRE at 25 as observed by Reem et al. (1999). Likely candidates for conferring cell specificity in response to PMA are the many PKC isozymes that have to date been described (Dekker and Parker, 1994). In Jurkat PMA stimulation results in the preferential activation of PKC-u and the activation of this PKC isoform leads to cooperations with other signaling molecules that do not occur when other PKC isozymes are activated (Werlen et al., 1998). Cell specificity of PKC responses can also be regulated by the composition of the AP-1 complex that is induced. The AP-1 complex is a heterodimer of Jun and Fos, which are members of the bZIP family of proteins. The many possible compositions of the AP-1 complex can selectively regulate gene expression (Mechta-Grigoriou et al., 2001). Watanabe et al. (2001) showed that in decidual cells the 1705 AP-1 site in the upstream PRL promoter preferentially binds AP-1 complexes that are composed of JunD and Fra-2. cAMP is a second messenger which can regulate the expression of target genes either in a positive or a negative manner. The effect of cAMP on a target gene is dependent on the transcription factors that are activated by PKA, and the signaling pathway that is elicited depends on cellspecific factors (Skalhegg and Tasken, 2000). PKA activation results in the phosphorylation of CREB/ATF proteins which can then interact with the transcription complex. In Jurkat cells, the PRL-1842-Luc construct was activated by cptcAMP. This cAMP responsiveness had been described earlier by Reem et al. (1999), who showed that in Jurkat cells a CRE located at 25 is in part responsible for cptcAMP induction of the upstream PRL promoter. We showed that cAMP also activates the upstream PRL promoter in IM-9, but not in K562 and U937 cells. In endometrial stromal cells, the upstream PRL promoter is also activated by cAMP but the activation in these cells is biphasic with a weak early induction apparent after 6 h and a strong delayed phase apparent after 24 h (Telgmann et al., 1997). Whereas the early activation is mediated by the CRE that is also used in Jurkat cells, the late response is mediated by two overlapping C/EBP sites in the region 332/ 270 (Pohnke et al., 1999). Although C/EBPs are probably not required for cAMP induction of the PRL promoter in Jurkat cells (Reem et al., 1999), the involvement of these transcription factors in the activation of the upstream PRL promoter in other leukemic cell lines and in primary leukocytes should be further investigated. We also showed that the cptcAMP – PMA synergism, described earlier in Jurkat cells using a construct with only 375 bp of the upstream PRL promoter (1999), was absent in all other cell lines. The cis- and trans- acting factors involved in this synergism remain to be elucidated. In addition, our results suggest that Ca2 + is not able to affect PRL-1842Luc activation on its own, but that it modulates transcrip-
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tional activation in combination with other signaling molecules. To compare promoter activation with endogenous PRL regulation, we assessed the effects of cptcAMP, PMA, and ionomycin on mRNA expression and protein secretion by Jurkat cells, because they produce high amounts of endogenous PRL. Since regulation of PRL mRNA and PRL secretion parallels the activation of the PRL-1842-Luc construct, we hypothesize that, in Jurkat, cptcAMP, PMA, and ionomycin modulate PRL expression and secretion through transcriptional regulation. The eosinophilic cell line Eol-1, which produces bioactive PRL, could not be transfected, and was used to study endogenous PRL expression. Since PRL expression in Eol-1 cells was only stimulated by cptcAMP, but not by PMA or ionomycin, we conclude that different cell lines also exhibit distinct activation mechanisms for endogenous PRL expression. Western blot analysis revealed that the secreted protein that was regulated in Jurkat and Eol-1 is 23 kDa PRL, which co-migrates with the major form of PRL produced and secreted by the pituitary. In Jurkat, secretion of a 25-kDa PRL immunoreactivity, probably representing a glycosylated variant that has also been described in the pituitary (Lewis et al., 1985; Markoff and Lee, 1987), was also induced. Although we show that PMA can stimulate PRL promoter activation in four different cell lines and endogenous PRL expression in Jurkat, PMA markedly inhibited basal PRL expression in PBMC from six out of seven donors. PMA only stimulated PRL expression in synergism with cptcAMP in PBMC from one donor. In contrast, cptcAMP stimulated PRL expression in PBMC from all donors, although the magnitude of the increase varied from 2- to 30-fold. Remarkably, in cells that exhibited the lowest response to cptcAMP, we observed the synergistic effect of cptcAMP and PMA. Possibly, alternative use of different signals to regulate PRL gene transcription is not only celltype specific, but also depends on the activation state of the cell. For instance, basal PKA and PKC activation may be influenced by subclinical infections or allergic reactions. Taken together, the effects of cptcAMP on promoter activation in Jurkat, and its effects on endogenous PRL expression in Jurkat, Eol-1, and PBMC, indicate that cAMP is an important regulator of PRL expression in the immune system. Although preliminary experiments in which we have separated T-cells from non-T-cells reveal that cptcAMP enhances PRL mRNA expression in both fractions, we cannot exclude that different regulation mechanism, as observed in K562 and U937 cells, are operative in certain mononuclear cell subpopulations. Likewise, these alternative mechanisms in myeloid cell lines may also be involved in the regulation of PRL expression in polymorphonuclear cells, which also express PRL mRNA (Kooijman et al., 2000a). Since cptcAMP stimulates endogenous PRL expression in leukemic cell lines and in normal PBMC, we inves-
tigated whether stimuli that signal through cAMP could also affect PRL expression in these cells. PGE2 is readily produced by activated monocytes and it is a common inflammatory mediator known to suppress Th1 responses and drive T-cell development towards the Th2 subset (Goodwin and Ceuppens, 1983; Fedyk et al., 1997). The effect of PGE2 on the activation of the PRL-1842-Luc construct in Jurkat (Fig. 8) was comparable to the effect of cptcAMP (Fig. 1). However, the stimulatory effect of PGE2 on endogenous PRL expression exceeded that of cptcAMP both at the mRNA and at the protein levels. A possible explanation for this discrepancy is the presence of enhancers, responding to PGE2, which are not contained in the PRL-1842-Luc construct. Furthermore, PGE2 could have an effect on PRL mRNA stability. We are presently investigating these possibilities. The importance of PGE2 in regulation of PRL expression in normal cells of the immune system is indicated by the stimulation of mRNA levels in PBMC as observed in four out of six donors. Whether differences in the response to cptcAMP and PMA are responsible for the variation in PGE2 responsiveness is not yet clear. PRL expression by normal PBMC was also enhanced by the h-adrenergic agonist terbutaline in three out of four donors, indicating that PRL expression in PBMC can also be modulated by catecholamines. Indeed, h-adrenergic receptors are expressed on T-cells, B-cells, NK-cells, and monocytes (Xu, 2001). Both PGE2 and catecholamines have been shown to influence autoimmune disease and PRL expression is upregulated in several autoimmune disorders. Enhanced PRL expression was found in PBMC from systemic lupus erythematosus patients and in salivary glandular epithelial cells of patients with Sjo¨gren syndrome (Larrea et al., 1997; Steinfeld et al., 2000). In patients with rheumatoid arthritis, synovial infiltrating T-cells secreted PRL which enhanced proliferation of synovial cells (Nagafuchi et al., 1999), suggesting local PRL might affect the course of inflammatory reactions. Further investigations are required to address the role of PGE2 and catecholamines in the regulation of PRL expression in leukocytes under normal and pathological conditions.
Acknowledgements This research was supported by the Flemish Government (GOA 97-02-04), the Fund for Scientific Research— Flanders (F.W.O. G0167.98N) and institutional grants from the VUB. Wim Vanden Berghe is a postdoctoral fellow with the Fund for Scientific Research—Flanders. We thank J. Schiettecatte for performing PRL assays, A. Stevens for the PRL-1842-Luc construct, J. Davis and R. Hooghe for helpful discussions and critical revision of the manuscript, B. Gellersen for the PRL3000-Luc construct and N.Y. Sinha for the VLS-2 antihPRL antibody.
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prolactin upon activation and differentiation of human B lymphocytes. J. Neuroimmunol. 47, 35 – 40. Larrea, F., Martinez-Castillo, A., Cabrera, V., Alcocer-Varela, J., Queipo, G., Carino, C., Alarcon-Segovia, D., 1997. A bioactive 60-kilodalton prolactin species is preferentially secreted in cultures of mitogenstimulated and nonstimulated peripheral blood mononuclear cells from subjects with systemic lupus erythematosus. J. Clin. Endocrinol. Metab. 82, 3664 – 3669. Lewis, U.J., Singh, R.N., Sinha, Y.N., Vanderlaan, W.P., 1985. Glycosylated human prolactin. Endocrinology 116, 359 – 363. Markoff, E., Lee, D.W., 1987. Glycosylated prolactin is a major circulating variant in human serum. J. Clin. Endocrinol. Metab. 65, 1102 – 1106. Matera, L., 1996. Endocrine, paracrine and autocrine actions of prolactin on immune cells. Life Sci. 59, 599 – 614. Matera, L., Contarini, M., Bellone, G., Forno, B., Biglino, A., 1999. Upmodulation of interferon-gamma mediates the enhancement of spontaneous cytotoxicity in prolactin-activated natural killer cells. Immunology 98, 386 – 392. Mechta-Grigoriou, F., Gerald, D., Yaniv, M., 2001. The mammalian Jun proteins: redundancy and specificity. Oncogene 20, 2378 – 2389. Montgomery, D.W., 2001. Prolactin production by immune cells. Lupus 10, 665 – 675. Nagafuchi, H., Suzuki, N., Kaneko, A., Asai, T., Sakane, T., 1999. Prolactin locally produced by synovium infiltrating T lymphocytes induces excessive synovial cell functions in patients with rheumatoid arthritis. J. Rheumatol. 26, 1890 – 1900. Pellegrini, I., Lebrun, J.J., Ali, S., Kelly, P.A., 1992. Expression of prolactin and its receptor in human lymphoid cells. Mol. Endocrinol. 6, 1023 – 1031. Perez, C.C., Penalva, R., Paez, P.M., Renner, U., Reul, J.M., Stalla, G.K., Holsboer, F., Arzt, E., 1999. Early activation of thyrotropin-releasinghormone and prolactin plays a critical role during a T cell-dependent immune response. Endocrinology 140, 690 – 697. Pohnke, Y., Kempf, R., Gellersen, B., 1999. CCAAT/enhancer-binding proteins are mediators in the protein kinase A-dependent activation of the decidual promoter. J. Biol. Chem. 27, 24808 – 24818. Reem, G.H., Ray, D.W., Davis, J.R., 1999. The human prolactin gene upstream promoter is regulated in lymphoid cells by activators of Tcells and by cAMP. J. Mol. Endocrinol. 22, 285 – 292. Sabharwal, P., Glaser, R., Lafuse, W., Varma, S., Liu, Q., Arkins, S., Kooijman, R., Kutz, L., Kelley, K.W., Malarkey, W.B., 1992. Prolactin synthesized and secreted by human peripheral blood mononuclear cells: an autocrine growth factor for lymphoproliferation. Proc. Natl. Acad. Sci. U. S. A. 89, 7713 – 7716. Skalhegg, B.S., Tasken, K., 2000. Specificity in the cAMP/PKA signaling pathway. Differential expression, regulation, and subcellular localization of subunits of PKA. Front. Biosci. 5, D678 – D693. Steinfeld, S., Rommes, S., Francois, C., Decaestecker, C., Maho, A., Appelboom, T., Heizmann, C.W., Kiss, R., Pochet, R., 2000. Big prolactin 60 kDa is overexpressed in salivary glandular epithelial cells from patients with Sjogren’s syndrome. Lab. Invest. 80, 239 – 247. Stevens, A., Ray, D.W., Worthington, J., Davis, J.R., 2001. Polymorphisms of the human prolactin gene—implications for production of lymphocyte prolactin and systemic lupus erythematosus. Lupus 10, 676 – 683. Telgmann, R., Maronde, E., Tasken, K., Gellersen, B., 1997. Activated protein kinase A is required for differentiation-dependent transcription of the decidual prolactin gene in human endometrial cells. Endocrinology 138, 929 – 937. Theas, M.S., De Laurentis, A., Lasaga, M., Pisera, D., Duvilanski, B.H., Seilcovich, A., 1998. Effect of lipopolysaccharide on tumor necrosis factor and prolactin release from rat anterior pituitary cells. Endocrine 8, 241 – 245. Theas, S., De Laurentiis, A., Candolfi, M., Lopez, S.L., Carrasco, A.E., Zaldivar, V., Seilicovich, A., 2001. Nitric oxide mediates the inhibitory effect of tumor necrosis factor-alpha on prolactin release. Neuroendocrinology 74, 82 – 86.
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Regulation of prolactin expression in leukemic cell lines and peripheral blood mononuclear cells Sarah Gerlo a,*, Wim Vanden Berghe b, Peggy Verdood a, Elizabeth L. Hooghe-Peters a, Ron Kooijman a a
Department of Pharmacology, Free University of Brussels (V.U.B.), Laarbeeklaan 103, B-1090 Brussels, Belgium b Department of Molecular Biology, University of Ghent, Ledeganckstraat 35, 9000 Ghent, Belgium Received 21 August 2002; received in revised form 25 November 2002; accepted 25 November 2002
Abstract To address the role of different intracellular signals in prolactin (PRL) expression in leukocytes, we have investigated the effects of chlorophenylthio-cAMP (cptcAMP), phorbol myristate acetate (PMA) and ionomycin on the activation of the upstream PRL promoter in several leukemic cell lines. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. In freshly isolated peripheral blood mononuclear cells (PBMC), PRL expression could only be stimulated by cptcAMP. The physiological importance of cAMP in the regulation of PRL expression in leukocytes is suggested by the finding that in PBMC, PRL expression is enhanced by prostaglandin-E2 and the h2-adrenergic agonist terbutaline, which both signal through cAMP. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Prolactin; Leukocytes; PMA; cAMP; Calcium; Prostaglandins
1. Introduction PRL is a 23-kDa polypeptide hormone that is produced mainly by the anterior lobe of the pituitary. The best known function of pituitary-derived PRL is the initiation and maintenance of lactation. However, PRL is a pleiotropic and versatile hormone that also plays a role in reproduction, angiogenesis and modulation of immune responses. Indeed, PRL receptors are widely distributed in a variety of tissues. Furthermore, several extrapituitary sources of PRL have been described and it is now well established that prolactin is expressed in the decidua, the brain, the mammary gland and in cells of the immune system (Ben Jonathan et al., 1996; Kooijman et al., 2000a). The fact that PRL subserves many different functions can, in part, be explained by tissuespecific regulation of PRL expression. Regulation of PRL expression in the pituitary has been extensively studied, but the regulation of ectopic PRL expression has remained relatively unexplored. The classical modulators of pituitary PRL expression [i.e. dopamine, thyrotropin releasing hormone (TRH) and estrogen] are unable to regulate extrapituitary PRL expression (Gellersen et al., 1989; Handwerger * Corresponding author. Tel.: +32-2-477-4462; fax: +32-2-477-4464. E-mail address: [email protected] (S. Gerlo).
et al., 1991; Bonhoff and Gellersen, 1994). This can be explained by the finding that in humans transcription in extrapituitary tissues is directed by an alternative promoter located 5.8 kb upstream of the pituitary-specific start site (Berwaer et al., 1994; Gellersen et al., 1994). Due to the presence of a 5Vnon-coding exon (exon 1a), the extrapituitary transcript of the PRL gene is 150 nucleotides longer than its pituitary counterpart. The PRL peptide produced by decidua and lymphocytes is, however, indistinguishable from that of pituitary origin. In the immune system, the PRL receptor, which belongs to the cytokine-receptor superfamily, is expressed on T-cells, B-cells, monocytes, and NK-cells (Pellegrini et al., 1992; Matera, 1996). Although PRL does not seem to be required for immune responses under normal circumstances in mice (Horseman et al., 1997), there is nevertheless compelling evidence for modulatory actions of PRL on both humoraland cell-mediated immune responses. For instance, PRL enhances the expression of inducible nitric oxide synthetase, immunoglobulins, and cytokines in human leukocytes (Dogusan et al., 2000; Jacobi et al., 2001; Lahat et al., 1993; Matera et al., 1999; Cesario et al., 1994). The immunomodulatory effects of PRL suggest that it plays a role in the communication between the neuroendocrine system and the immune system. Indeed, pituitary PRL secretion is regulated
0165-5728/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. doi:10.1016/S0165-5728(02)00438-1
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by endotoxin, cytokines and during T-cell-dependent immune responses (Theas et al., 1998, 2001; Perez et al., 1999). Furthermore, local effects of PRL in the immune system are suggested by the expression of PRL in many different types of leukocytes. For instance, PRL mRNA was found to be expressed in thymocytes, T-cells, and B-cells (Pellegrini et al., 1992; Montgomery, 2001). Whereas leukocyte-derived PRL may be involved in normal immune responses, the regulation of PRL expression in leukocytes has hardly been addressed. In peripheral blood mononuclear cells (PBMC), PRL expression is stimulated by phytohaemagglutinin (PHA) and concanavalin A (Con A) (20 –21). Reem et al. (1999) have shown that in the leukemic T-cell line Jurkat, the upstream PRL promoter is activated by cptcAMP, a long-lasting cAMP analogue, and that this activation is synergistically influenced by the phorbolester PMA. A CRE at 25 bp in the upstream PRL promoter is partially responsible for this induction. Additionally, enhanced PRL production in leukocytes has been observed in patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and in some patients with acute myeloid leukemia (AML) (Larrea et al., 1997; Gutierrez et al., 1995; Kooijman et al., 2000b). It has furthermore been suggested that PRL produced by leukemic blasts can contribute to enhanced serum PRL concentrations (Hatfill et al., 1990; Ales and Byrd, 2001). These findings indicate that modulation of extrapituitary PRL expression might play a role in the pathophysiology of immune and hematological disorders and prompted the present investigations on the regulation of PRL expression in the immune system. The present study was designed to further investigate the regulation of PRL in different leukemic cells and in PBMC from normal donors. To address intracellular signals leading to promoter activation, we have studied the effects of PMA, cAMP, and the Ca2 + ionophore ionomycin in leukemic cells that were transfected with reporter plasmids carrying the upstream PRL promoter. All three stimulators, alone or in synergism with each other, were able to modulate promoter activity, but their actions were cell-type dependent. The effects on promoter activation in transfected Jurkat cells reflected regulation of endogenous PRL expression. In the PBMC, only cAMP stimulated PRL gene expression, whereas PMA and ionomycin were inhibitory. A similar response was found in the PRL secreting myeloid cell line Eol-1. We furthermore show that extracellular stimuli, which use cAMP as a second messenger (PGE2 and the h2-agonist terbutaline), enhance PRL expression in PBMC.
antiserum VLS-2 was donated by Y.N. Sinha (San Diego, CA). Human PRL cDNA was provided by J. Martial (Lie`ge, Belgium). The human T-leukemic cell line Jurkat, the human B-lymphoblastoid cell line IM-9, and the myeloid leukemic cell lines Eol-1, K562, and U937 were obtained from the European Collection of Cell Cultures (Salisbury, UK). PGE2, cptcAMP, ionomycin, PGE2, and terbutaline were purchased from Calbiochem (Darmstadt, Germany). DEAE-dextran and PMA were from Sigma (Bornem, Belgium). Recombinant hPRL was produced in Escherichia coli in our lab using a cDNA obtained from J. Martial (Lie`ge, Belgium). GF109203X was purchased from Biomol (Plymouth, PA). 2.2. Subjects Blood donors were between 25 and 55 years of age. Informed consent was obtained from all blood donors and the research protocol has been approved by the local ethical committee. 2.3. Cell preparation and cell culture Human PBMC were purified from heparinized venous blood drawn from healthy donors. PBMC were isolated by centrifugation on Ficoll-Isopaque (Pharmacia & Upjohn, Uppsala, Sweden) density gradients (1.077 g/ml) at 1000 g for 20 min at room temperature and subsequently resuspended in RPMI at a concentration of 107/ml. Cells were cultured in Falcon polystyrene six-well plates (Beckton Dickinson Labware Europe, France). Jurkat, IM-9, and Eol-1 cells were maintained in RPMI supplemented with 10% FCS, K562, and U937 cells in RPMI supplemented with 10% NBCS. For stimulation experiments, cell lines were resuspended in RPMI at a concentration of 2 106/ ml and cultured in Falcon polystyrene 6- or 24-well plates (Beckton Dickinson Labware). All cell cultures were done in a humidified 5% CO2 atmosphere at 37 jC. To assess regulation of prolactin expression, cells were cultured with the appropriate stimuli in RPMI for 18 h. PMA, cptcAMP, and ionomycin were used at concentrations of 10 ng/ml, 250 AM, or 200 ng/ml, respectively. cptcAMP was dissolved in ddH2O, PMA, and ionomycin in dimethylsulfoxide (DMSO). In all conditions, the final DMSO concentration never exceeded 0.02% and was without effect. 2.4. Plasmid constructs
2. Materials and methods 2.1. Reagents RMPI 1640 (with glutamax), newborn calf serum (NBCS), and fetal calf serum (FCS) were purchased from Life Technologies (Merelbeke, Belgium). Anti-hPRL rabbit
The plasmid construct PRL-1842-Luc, containing the region between 1842 and + 35 of the upstream PRL promoter inserted into the pGL3 enhancer reporter plasmid (Promega, Madison, WI), was obtained from A. Stevens and J. Davis (Manchester, UK). Deletion constructs were prepared using fragments of the 1842/ + 35 region that were generated by digestion with restriction enzymes. The
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pGL3 enhancer vector was linearized using SmaI and NcoI and restriction fragments with appropriately modified ends were inserted by semi-blunt ligation. The PRL-3000Luc construct was a gift from B. Gellersen (Hamburg, Germany). 2.5. Transient transfections Jurkat, U937, IM-9, and K562 cells were transfected by electroporation. Cells growing in the log phase were washed and resuspended in RPMI at a concentration of 20 106 cells/ml. Plasmids were added to a concentration of 20 Ag/ml and electroporation was performed using the following settings: 330 V and 1800 AF for Jurkat, 300 V and 1800 AF for U937 and IM-9, and 300 V and 1500 AF for K562 (Electropore 2000, ‘‘home-made’’ device, laboratory of J. Martial, Lie`ge, Belgium). Following electroporation, cells were resuspended in culture medium. After a recovery period of 24 h, cells were washed and resuspended in RPMI at a concentration of 1 106/ml. Subsequently, cells were cultured in 24-well plates (0.5 ml/ well) for 18 h in the presence of the appropriate stimuli. Cell lysates were prepared and assayed for luciferase activity using the Promega Luciferase Assay kit (Promega), following the manufacturer’s instructions. Luminescence was measured using a Biocounter M 1500 luminometer (Celsis, Landgraaf, The Netherlands). Data were calculated as luciferase activity per milligram protein (BioRad protein assay, Bio-Rad, Munich, Germany) and were expressed as fold induction relative to the untreated samples.
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2.8. Western blotting Conditioned media were concentrated 50 times using Ultrafree MC concentrators (Millipore, Bedford, MA), after which protein content was determined using the Bio-Rad protein assay (Bio-Rad). Equal amounts of protein were subjected to polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking for 1 h in blocking buffer (0.05% Tween20 and 2% non-fat dry milk in PBS), blots were incubated with the rabbit anti-PRL serum VLS-2 (1:4000 in blocking buffer) for 18 h at 4 jC. Subsequently, blots were washed three times for 10 min in PBS/0.5% Tween-20 and incubated with a peroxidase-conjugated goat anti-rabbit IgG antiserum (1:10,000 in blocking buffer). After three more washes, antigen – antibody complexes were detected using Western Lightning detection reagent (Perkin Elmer Life Sciences, Boston, MA) according to the manufacturer’s instructions. 2.9. Statistical analysis Statistical differences between group means were determined by ANOVA with Tukey’s post-test. Differences were considered significant when p < 0.05. Data represented are means F S.D. of at least three independent experiments.
3. Results 3.1. Activation of the upstream PRL promoter by PMA, cptcAMP, and ionomycin
2.6. Reverse transcription-PCR and Southern blotting Total RNA was isolated using Trizol reagent according to the manufacturer’s instructions (Gibco-BRL). For PCR, we used a sense primer corresponding to a sequence within exon 1a (5V-GAG-ACA-CCA-AGA-AGA-ATC-GGA-3V) and an antisense primer corresponding to a sequence in exon 5 (5V-ATG-ATT-CGG-CAC-TTC-AGG-AGC-3V). Reverse transcription was performed as described earlier (Dogusan et al., 2001). For PCR amplification of PRL cDNA, reaction mixtures were subjected to 35 cycles of PCR (denaturation at 94 jC for 60 s, annealing at 55 jC for 60 s, and extension at 72 jC for 90 s). PCR for GADPH was performed as described earlier (Dogusan et al., 2001). 2.7. PRL assays PRL concentrations in conditioned media were determined using an electrochemiluminescence immunoassay (ECLIA; Elecsys 1010/2010 Systems) from Roche Diagnostics (Manheim, Germany). Monoclonal antibodies used in this sandwich ELISA do not cross-react with hGH, hHCG, hPL, TSH, FSH, or LH. The detection limit of this assay is 0.5 ng/ml.
Promoter activation was assessed using leukemic cell lines that were transiently transfected with a PRL-1842-Luc construct. As depicted in Fig. 1, PMA stimulates promoter activation in the Jurkat T-cell line, the IM-9 B-lymphoblastoid cell line, the K562 erythroblastoid cell line, and the myelomonocytic cell line U937. Since PMA is known to stimulate protein kinase C (PKC) activity, we assessed the effects of PMA in the presence of the PKC inhibitor GF109203X, a bisindolylmaleimide with high specificity for PKC as compared to other protein kinases (Toullec et al., 1991). Stimulation of promoter activity in Jurkat, K562, U937, and IM-9 cells was blocked by this inhibitor, whereas basal promoter activity was not affected (Fig. 1). Deletion of sequences 1842/ 1662 or 1842/ 967 of our promoter construct resulted in the disappearance of PMA effects in K562, U937, and IM-9 cells but not in Jurkat cells (Fig. 2), indicating the presence of a ‘‘PMA responsive element’’ in the distal region between coordinates 1842 and 1662, which is operative in K562, U937, and IM-9 cells and of another ‘‘PMA responsive element’’ between 967 and + 35, which is operative in Jurkat. In Jurkat cells, the cAMP analogue cptcAMP stimulates promoter activation by 2-fold in the absence of other factors,
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Fig. 1. PMA induces activation of PRL-1842-Luc in several leukemic cell lines and this activation is inhibited by the PKC inhibitor GF109203X. Jurkat, IM-9, K562, and U937 cells were transiently transfected with PRL1842-Luc. Cells were preincubated for 1 h with 1 AM (for Jurkat, K562, and U937) or 10 AM (IM-9) GF109203X, before stimulation with 10 ng/ml PMA. After 18 h, cells were harvested. Reporter gene activation is expressed as fold induction relative to unstimulated control values. PMA promoter activation (versus control) and inhibition of PMA activation by GF109203X were significant ( p < 0.05) in all cell lines.
and exerts a strong synergistic effect with PMA (Fig. 3). These results are in accordance with the results of Reem et al. (1999) using Jurkat cells transfected with a 375Luc PRL promoter construct. The synergistic effects of cptcAMP and PMA were still observed in Jurkat cells transfected with 1662, 1519, or 967-Luc constructs (data not shown). cptcAMP also stimulated PRL-1842-Luc in IM-9 cells by 3-fold, but in these cells PMA and cptcAMP have additive rather than synergistic effects. Remarkably, no effects of cptcAMP were observed in K562 and U937 cells (Fig. 3). The Ca2 + ionophore ionomycin alone did not affect basal promoter activity, but interfered with promoter activation by PMA or cptcAMP. In Jurkat cells, ionomycin strongly augmented promoter activation in synergism with cptcAMP. The cptcAMP effect in IM-9 cells was unaffected by ionomycin. Both in Jurkat and
Fig. 2. Effects of PMA on activation of PRL-1842-Luc constructs with 5V deletions. Jurkat, IM-9, K562, and U937 cells were transiently transfected with equimolar amounts of PRL-1842-Luc, PRL-1662-Luc, PRL-1519Luc, and PRL-967-Luc. Reporter gene activation is expressed as fold induction relative to unstimulated control values. The effects of PMA on constructs with 5Vdeletions were significantly ( p < 0.05) different from PMA activation of the PRL-1842-Luc construct in all cell lines.
Fig. 3. Cell-specific activation of the upstream PRL promoter by PMA, cptcAMP, and ionomycin. Jurkat, IM-9, K562, and U937 cells were transiently transfected with PRL-1842-Luc. Reporter gene activation is expressed as fold induction relative to unstimulated control values. (a) Different from unstimulated control with p < 0.001; (b) Different from parallel condition without ionomycin with p < 0.001; (c) Different from parallel condition without ionomycin with p < 0.05.
IM-9 cells, the effect of the combination of PMA, cptcAMP, and ionomycin on promoter activation was smaller than that of PMA and cptcAMP in IM-9, or that of cptcAMP with either PMA or ionomycin in Jurkat cells. Furthermore,
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endogenous PRL. As we have chosen the sense PCR primer in exon 1a, we have only amplified the long, decidual PRL (dPRL) message and not the short, pituitary PRL mRNA. Fig. 4A shows that PRL mRNA levels were moderately stimulated by PMA or cptcAMP. Combinations of cptcAMP with either PMA or ionomycin induced a strong stimulation of mRNA expression, which is in accordance with a strong induction of promoter activation in transfection studies (Fig. 1). Also in accordance with promoter studies is the smaller stimulation of mRNA expression when all three stimuli are added. Next, we studied the effects on PRL secretion by measuring PRL levels in conditioned medium after an 18-h culture period. We found a small stimulation of PRL secretion by either PMA or cptcAMP, and a strong stimulation by cptcAMP in combination with either PMA or ionomycin (Fig. 4B). Since the production of immunoreactive PRL variants by leukocytes has been described in various studies (Sabharwal et al., 1992; Larrea et al.,
Fig. 4. Regulation of PRL expression in Jurkat by PMA, cptcAMP, and ionomycin. (A) Regulation at the mRNA level as assessed by RT-PCR. PCR amplified products were subjected to agarose gel electrophoresis. Densitometric scanning analysis was performed on ethidium bromide stained gels and PRL mRNA quantity was corrected for differences in GADPH mRNA content. The histogram represents the mean values from three independent experiments, whereas the gels are representative of three independent experiments. (B) Regulation of PRL secretion as assessed by ECLIA. (C) Detection of 23 kDa PRL in conditioned media by Western blotting; 23 kDa PRL was not detected when normal rabbit serum was used (not shown). Specificity of the binding of anti-PRL antiserum was confirmed by preincubation of the antiserum with 0.1 Ag of rhPRL for 1 h at 37 jC. The data in B and C represent two different experiments.
ionomycin slightly inhibits PMA promoter activation in K562 and IM-9, but not in Jurkat and U937 cells (Fig. 3). All stimuli were used at concentrations that did not affect cell viability as assessed by trypan blue exclusion. 3.2. Regulation of endogenous PRL in cell lines To address whether the effects of cptcAMP, PMA, and ionomycin on promoter activation are representative for regulation of endogenous PRL, we investigated the effects of these stimulators on PRL mRNA expression and PRL secretion by Jurkat cells, because this cell line expresses
Fig. 5. Regulation of PRL expression in Eol-1 by PMA, cptcAMP, and ionomycin. (A) Regulation of mRNA, assessed and quantified as described in Fig. 4. (B) Regulation of PRL secretion as assessed by ECLIA. (C) Detection of 23 kDa PRL in conditioned media by Western blotting as described in Fig. 4. The data in B and C represent two different experiments.
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1997), we assessed the molecular weight of immunoreactive PRL that was secreted upon stimulation with PMA, cptcAMP, or ionomycin by Western blot analysis. We found that PMA or cptcAMP alone weakly stimulates the expression of 23 kDa PRL, whereas the combinations of cptcAMP with PMA or ionomycin strongly activate 23 kDa PRL and also weakly induce the secretion of 25 kDa PRL (Fig. 4C). Taken together, these results indicate that promoter activation in Jurkat parallels mRNA expression and the secretion of 23 kDa PRL. To further address endogenous regulation of PRL in leukocytes, we investigated regulation of mRNA levels and protein secretion in another leukemic (eosinophilic) cell line, Eol-1, that expresses high levels of PRL (Kooijman et al., 2000b). Activation studies using this cell line revealed that cptcAMP markedly increased PRL mRNA levels, whereas PMA and ionomycin were without effect (Fig. 5A). Addition of PMA slightly reduced the stimulation by cptcAMP, whereas the presence of ionomycin completely abrogated the effect of cptcAMP. Protein studies revealed that PRL secretion generally reflected mRNA levels and was mainly stimulated by cptcAMP alone (Fig. 5B). In addition, Western blot analysis showed that cptcAMP only stimulated the secretion of 23 kDa PRL (Fig. 5C). Thus, in Eol-1 cells, mRNA levels and secretion of 23 kDa PRL were mainly regulated by cAMP. However, promoter activation in Eol-1 cells could not be established, because we have been so far unable to transfect these cells efficiently.
Fig. 7. Regulation of PRL mRNA expression by PGE2 and terbutaline in normal PBMC. Expression of mRNA was measured by RT-PCR, followed by Southern blotting. The results are representative of donors that responded to PGE2 (4 out of 6) and terbutaline (3 out of 4).
whereas PMA always reduced basal mRNA levels. Remarkably, the effect of cptcAMP was completely inhibited by PMA in six out of seven donors, whereas a synergistic effect of cptcAMP and PMA was observed in one donor (Fig. 6C). Notably, the magnitude of the stimulating effects of cptcAMP were variable (Fig. 6A vs. B) and the synergistic effect was observed in the donor with the smallest response to cptcAMP alone (Fig. 6C). The effects of ionomycin were assayed in PBMC from four donors, and it appeared that ionomycin, alone or in combination with either PMA or cptcAMP, inhibited PRL expression (Fig. 6A: 5 –8).
3.3. Regulation of PRL expression in normal human PBMC To assess the regulation of PRL expression in normal leukocytes, we analyzed the effects of cptcAMP, PMA, and ionomycin on freshly isolated PBMC from seven normal donors. We found that cptcAMP increased PRL mRNA expression in PBMC from all seven donors tested (Fig. 6),
Fig. 6. Regulation of PRL mRNA expression in PBMC. Expression of mRNA was measured by RT-PCR, followed by Southern blotting. Characteristic responses to stimulation with PMA (2), cptcAMP (3), and PMA + cptcAMP (4) as compared to the unstimulated control (1) are represented for three donors (A, B, and C). The effect of ionomycin (5), ionomycin + PMA (6), ionomycin + cptcAMP (7), or ionomycin + PMA + cptcAMP (8), was similar in all donors tested and is shown for donor A.
Fig. 8. Regulation of PRL promoter activation and endogenous PRL expression by PGE2 in Jurkat. Jurkat cells were stimulated for 18 h with 1, 10, and 100 nM PGE2. (A) Jurkat cells transiently transfected with PRL1842-Luc. (B) PCR amplified products were subjected to agarose gel electrophoresis and visualised by ethidium bromide staining. (C) PRL secretion as assessed by ECLIA.
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3.4. Stimulation of PRL expression by PGE2 and the 2adrenergic receptor agonist terbutaline The finding that cAMP is a major regulator of PRL mRNA expression in PBMC and some leukemic cell lines prompted us to study the effects of physiological modulators of the immune system which operate through cAMP as a second messenger. PGE2 has been implicated in the regulation of both cellular and humoral immune responses (Goodwin and Ceuppens, 1983) and operates via cAMP and other second messengers. To test the possible effects of h-adrenergic receptors, which contribute to neuromodulation of immune responses (Xu, 2001), we incubated cells with the h2-adrenergic receptor agonist terbutaline. As shown in Fig. 7, 10 –100 AM PGE2 increased PRL mRNA levels in PBMC of four out of six donors, whereas 1 – 10 AM terbutaline did so in three out of four donors. In Jurkat cells, terbutaline had no effect on PRL expression (data not shown), whereas PGE2 increased PRL mRNA levels (Fig. 8B). In Jurkat cells, PGE2 also stimulated promoter activation (Fig. 8A) and induced a concomitant increase in PRL secretion (Fig. 8C).
4. Discussion The regulation of prolactin expression in leukocytes is different from that seen in the pituitary. The basis of this divergent cell-specific transcription appears to be the use of an alternative promoter in extrapituitary tissues such as the decidua and leukocytes (Berwaer et al., 1994; Gellersen et al., 1994). In the present study, we show that a promoter construct, carrying 1842 bp of the upstream PRL promoter, was activated by PMA through activation of PKC in all leukocytic cell lines we investigated. Reem et al. (1999) did not find activation of the upstream PRL promoter with PMA alone in Jurkat, which can be explained by the fact that they used a construct carrying only 375 bp of the upstream promoter. Indeed, more recently, they showed PMA induction of upstream PRL promoter activity in Jurkat using the PRL-1842-Luc construct (Stevens et al., 2001). The observations of Reem et al. (1999) and our finding that PMA responsiveness is retained with a 967-Luc PRL promoter construct, suggest that another PMA responsive element is located between 967 and 375 of the upstream PRL promoter. In K562, U937, and IM-9 cells, the responsive element for the PMA effect is localised between 1842 and 1662 in the upstream PRL promoter. Watanabe et al. (2001) have described an AP-1 site at 1705 in the upstream PRL promoter that appeared to function as an enhancer in decidualised endometrial stromal cells but not in non-decidual cells. The authors did not find activation of this enhancer region when it was transfected into Jurkat cells, nor did they find a footprint using Jurkat cell nuclear extract. Our findings support the idea that the 1705 AP-1 site in the upstream PRL promoter is not functional in Jurkat
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cells. However, it could play a role in other leukemic cells such as K562, U937, and IM-9 cells. In Jurkat cells, a third responsive element in the region 375/ + 35 acts synergistically with a CRE at 25 as observed by Reem et al. (1999). Likely candidates for conferring cell specificity in response to PMA are the many PKC isozymes that have to date been described (Dekker and Parker, 1994). In Jurkat PMA stimulation results in the preferential activation of PKC-u and the activation of this PKC isoform leads to cooperations with other signaling molecules that do not occur when other PKC isozymes are activated (Werlen et al., 1998). Cell specificity of PKC responses can also be regulated by the composition of the AP-1 complex that is induced. The AP-1 complex is a heterodimer of Jun and Fos, which are members of the bZIP family of proteins. The many possible compositions of the AP-1 complex can selectively regulate gene expression (Mechta-Grigoriou et al., 2001). Watanabe et al. (2001) showed that in decidual cells the 1705 AP-1 site in the upstream PRL promoter preferentially binds AP-1 complexes that are composed of JunD and Fra-2. cAMP is a second messenger which can regulate the expression of target genes either in a positive or a negative manner. The effect of cAMP on a target gene is dependent on the transcription factors that are activated by PKA, and the signaling pathway that is elicited depends on cellspecific factors (Skalhegg and Tasken, 2000). PKA activation results in the phosphorylation of CREB/ATF proteins which can then interact with the transcription complex. In Jurkat cells, the PRL-1842-Luc construct was activated by cptcAMP. This cAMP responsiveness had been described earlier by Reem et al. (1999), who showed that in Jurkat cells a CRE located at 25 is in part responsible for cptcAMP induction of the upstream PRL promoter. We showed that cAMP also activates the upstream PRL promoter in IM-9, but not in K562 and U937 cells. In endometrial stromal cells, the upstream PRL promoter is also activated by cAMP but the activation in these cells is biphasic with a weak early induction apparent after 6 h and a strong delayed phase apparent after 24 h (Telgmann et al., 1997). Whereas the early activation is mediated by the CRE that is also used in Jurkat cells, the late response is mediated by two overlapping C/EBP sites in the region 332/ 270 (Pohnke et al., 1999). Although C/EBPs are probably not required for cAMP induction of the PRL promoter in Jurkat cells (Reem et al., 1999), the involvement of these transcription factors in the activation of the upstream PRL promoter in other leukemic cell lines and in primary leukocytes should be further investigated. We also showed that the cptcAMP – PMA synergism, described earlier in Jurkat cells using a construct with only 375 bp of the upstream PRL promoter (1999), was absent in all other cell lines. The cis- and trans- acting factors involved in this synergism remain to be elucidated. In addition, our results suggest that Ca2 + is not able to affect PRL-1842Luc activation on its own, but that it modulates transcrip-
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tional activation in combination with other signaling molecules. To compare promoter activation with endogenous PRL regulation, we assessed the effects of cptcAMP, PMA, and ionomycin on mRNA expression and protein secretion by Jurkat cells, because they produce high amounts of endogenous PRL. Since regulation of PRL mRNA and PRL secretion parallels the activation of the PRL-1842-Luc construct, we hypothesize that, in Jurkat, cptcAMP, PMA, and ionomycin modulate PRL expression and secretion through transcriptional regulation. The eosinophilic cell line Eol-1, which produces bioactive PRL, could not be transfected, and was used to study endogenous PRL expression. Since PRL expression in Eol-1 cells was only stimulated by cptcAMP, but not by PMA or ionomycin, we conclude that different cell lines also exhibit distinct activation mechanisms for endogenous PRL expression. Western blot analysis revealed that the secreted protein that was regulated in Jurkat and Eol-1 is 23 kDa PRL, which co-migrates with the major form of PRL produced and secreted by the pituitary. In Jurkat, secretion of a 25-kDa PRL immunoreactivity, probably representing a glycosylated variant that has also been described in the pituitary (Lewis et al., 1985; Markoff and Lee, 1987), was also induced. Although we show that PMA can stimulate PRL promoter activation in four different cell lines and endogenous PRL expression in Jurkat, PMA markedly inhibited basal PRL expression in PBMC from six out of seven donors. PMA only stimulated PRL expression in synergism with cptcAMP in PBMC from one donor. In contrast, cptcAMP stimulated PRL expression in PBMC from all donors, although the magnitude of the increase varied from 2- to 30-fold. Remarkably, in cells that exhibited the lowest response to cptcAMP, we observed the synergistic effect of cptcAMP and PMA. Possibly, alternative use of different signals to regulate PRL gene transcription is not only celltype specific, but also depends on the activation state of the cell. For instance, basal PKA and PKC activation may be influenced by subclinical infections or allergic reactions. Taken together, the effects of cptcAMP on promoter activation in Jurkat, and its effects on endogenous PRL expression in Jurkat, Eol-1, and PBMC, indicate that cAMP is an important regulator of PRL expression in the immune system. Although preliminary experiments in which we have separated T-cells from non-T-cells reveal that cptcAMP enhances PRL mRNA expression in both fractions, we cannot exclude that different regulation mechanism, as observed in K562 and U937 cells, are operative in certain mononuclear cell subpopulations. Likewise, these alternative mechanisms in myeloid cell lines may also be involved in the regulation of PRL expression in polymorphonuclear cells, which also express PRL mRNA (Kooijman et al., 2000a). Since cptcAMP stimulates endogenous PRL expression in leukemic cell lines and in normal PBMC, we inves-
tigated whether stimuli that signal through cAMP could also affect PRL expression in these cells. PGE2 is readily produced by activated monocytes and it is a common inflammatory mediator known to suppress Th1 responses and drive T-cell development towards the Th2 subset (Goodwin and Ceuppens, 1983; Fedyk et al., 1997). The effect of PGE2 on the activation of the PRL-1842-Luc construct in Jurkat (Fig. 8) was comparable to the effect of cptcAMP (Fig. 1). However, the stimulatory effect of PGE2 on endogenous PRL expression exceeded that of cptcAMP both at the mRNA and at the protein levels. A possible explanation for this discrepancy is the presence of enhancers, responding to PGE2, which are not contained in the PRL-1842-Luc construct. Furthermore, PGE2 could have an effect on PRL mRNA stability. We are presently investigating these possibilities. The importance of PGE2 in regulation of PRL expression in normal cells of the immune system is indicated by the stimulation of mRNA levels in PBMC as observed in four out of six donors. Whether differences in the response to cptcAMP and PMA are responsible for the variation in PGE2 responsiveness is not yet clear. PRL expression by normal PBMC was also enhanced by the h-adrenergic agonist terbutaline in three out of four donors, indicating that PRL expression in PBMC can also be modulated by catecholamines. Indeed, h-adrenergic receptors are expressed on T-cells, B-cells, NK-cells, and monocytes (Xu, 2001). Both PGE2 and catecholamines have been shown to influence autoimmune disease and PRL expression is upregulated in several autoimmune disorders. Enhanced PRL expression was found in PBMC from systemic lupus erythematosus patients and in salivary glandular epithelial cells of patients with Sjo¨gren syndrome (Larrea et al., 1997; Steinfeld et al., 2000). In patients with rheumatoid arthritis, synovial infiltrating T-cells secreted PRL which enhanced proliferation of synovial cells (Nagafuchi et al., 1999), suggesting local PRL might affect the course of inflammatory reactions. Further investigations are required to address the role of PGE2 and catecholamines in the regulation of PRL expression in leukocytes under normal and pathological conditions.
Acknowledgements This research was supported by the Flemish Government (GOA 97-02-04), the Fund for Scientific Research— Flanders (F.W.O. G0167.98N) and institutional grants from the VUB. Wim Vanden Berghe is a postdoctoral fellow with the Fund for Scientific Research—Flanders. We thank J. Schiettecatte for performing PRL assays, A. Stevens for the PRL-1842-Luc construct, J. Davis and R. Hooghe for helpful discussions and critical revision of the manuscript, B. Gellersen for the PRL3000-Luc construct and N.Y. Sinha for the VLS-2 antihPRL antibody.
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