Mol. Hum. Reprod. Advance Access originally published online on February 18, 2005
Molecular Human Reproduction 2005 11(4):237-243; doi:10.1093/molehr/gah149
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Ligand activated relaxin receptor increases the transcription of IGFBP-1 and prolactin in human decidual and endometrial stromal cells
1Department of Obstetrics and Gynecology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8091, USA
2 To whom correspondence should be addressed. E-mail: litseng{at}notes.cc.sunysb.edu
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Abstract |
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The aim of this study was to investigate relaxin (RLX) receptor-mediated gene activation in human endometrium. We determined the promoter activities of insulin-like growth factor binding protein-1 (IGFBP-1) and prolactin (PRL) and identified sequence(s) that mediate RLX activated transcription in human decidual cells and endometrial stromal cells. In human decidual cells, the promoter activity of IGFBP-1 was increased significantly in cells incubated with RLX. In endometrial stromal cells, the RLX mediated activation was enhanced only when stromal cells were co-transfected with RLX-receptor (LGR7) expression vector and RLX alone had little effect (Mazella et al., 2004
). Deletion and mutation analysis showed that the cAMP regulatory element (CRE, –263 to –259 bp) in the IGFBP-1 promoter was essential for the activation. In addition, RLX increased the phosphorylation of CRE binding protein (CREB to p-CREB) and p-CREB resided in the nucleus, indicating that RLX activates the protein kinase (PKA) system in decidual cells. Gel shift assay showed that nuclear extracts prepared from RLX treated decidual cells increased the binding to the CRE site of the IGFBP-1 promoter. RLX increased the PRL promoter activity mediated through the region containing multiple CCAAT/enhancer-binding proteins (C/EBP) binding sites that have been shown to mediate the PRL gene activation by cAMP analogue (Pohnke et al., 1999). RLX enhanced IGFBP-1 promoter activity was inhibited by cAMP dependent PKA inhibitor, H-89. PRL promoter activity was inhibited by both H-89 and U0126 indicating multiple signalling pathways are activated by RLX in endometrial cells for different target gene activation. |
Introduction |
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Progesterone induces decidualization of endometrium in the late luteal phase of the menstrual cycle. However, progesterone alone does not achieve morphological changes exhibited in decidua. In addition, progesterone is a rather weak stimulator for the activation of decidual cell marker proteins, prolactin (PRL) and insulin-like growth factor binding protein-1 (IGFBP-1). In vitro, relaxin (RLX) enhances progestin induced stromal cell differentiation and activation of IGFBP-1 and PRL genes (Tseng et al., 1992
; Lane et al., 1994). RLX in combination with phosphodiesterase (PDE) inhibitor has similar effects as RLX/progestin on the production of PRL and IGFBP-1 in endometrial stromal cells (Zhu et al., 1990; Bell et al., 1991; Bartsch et al., 2004) indicating that PDE may play a role in RLX-induced gene activation. Other factors, such as PGE2 (prostaglandin E), HCG, FSH, LH and cAMP generating agents also synergistically enhance progestin induced IGFBP-1 and PRL (Tang and Gurpide, 1993; Tang et al., 1993; Frank et al., 1994; Telgmann et al., 1997; Bartsch et al., 2004). The effect of RLX appears to be highly effective for endometrial cell differentiation and gene activation (Lane et al., 1994). Previously, we have shown that 1–2 ng/ml RLX, similar to the plasma levels in early pregnancy (Emmi et al., 1991), is sufficient to sustain the stimulation of IGFBP-1 and PRL. In addition, we and others have shown that the RLX is produced in human endometrial/decidual cells (Hansell et al., 1991; Palejwala et al., 2002). Therefore, RLX could be the major endogenous factor that promotes decidualization and induction of IGFBP-1 and PRL genes in endometrial stromal/decidual cells.
RLX receptor is known to be one of the G-protein coupled receptors, capable of mediating the action of RLX through a cAMP dependent pathway (Hsu et al., 2002). We have shown that the RLX receptor is increased by progestin in stromal cells (Tseng et al., 1992; Mazella et al., 2004). Thus, the synergistic effect of progestin and RLX may be derived from increased capacity of RLX receptor G-protein coupling activities. In fact, induction of IGFBP-1 is achieved by RLX alone in decidual cells that contain much higher levels of RLX receptor (Mazella et al., 2004). The RLX receptor activated genes and sequences that mediate the transcription of IGFBP-1 and PRL genes remain to be determined. In this study, we show that the RLX-induced phosphorylation of cAMP regulatory element binding protein (CREB) in decidual cells enhanced the binding to the cAMP regulatory element (CRE) site to promote the transcription of IGFBP-1. In addition, we found that RLX activated transcription of PRL was mediated through multiple CCAAT/enhancer-binding proteins (C/EBP) binding sites that have been shown to mediate PRL gene activation by cAMP analogue (Pohnke et al., 1999).
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Materials and methods |
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Endometrial cell culture
Human endometrial specimens were obtained from women of reproductive age (35–50 years old, still having menstrual cycles) who were undergoing hysterectomy for various medical reasons, but not related to any abnormality of the endometrium. In addition, decidual tissue samples were obtained from the decidual layer attached to the fetal membranes at the term of gestation. Permission for using these human specimens was approved by the Human Subject Committee of our institution in accordance with US Department of Health regulations.
Stromal cells (>95% purity) were isolated from endometrium by collagenase digestion as described previously (Zhu et al., 1990; Lane et al., 1994). Newly formed decidual cells, also called decidual fibroblasts (Richards et al., 1995; Brar et al., 2002) capable of attaching to the Petri dish surface, were isolated from the decidual layer of fetal membrane as previously described (Mazella et al., 2004). These decidual cells are capable of producing PRL and IGFBP-1 and they are also able to respond to hormone stimulation (Mazella et al., 2004). Isolated decidual cells (
95% purity) were identified by positive immunohistochemical staining for vimentin, PRL and IGFBP-1 (Zhu et al., 1990; Lane et al., 1994; Tseng et al., 1999; Palejwala et al., 2002). Cells isolated from a single specimen were cultured separately (no mixed culture from different specimens). To study the effect of RLX, culture medium (RPMI 1640 medium with 2% charcoal–dextran stripped fetal bovine serum) was supplemented with porcine RLX (Sherwood et al., 1993). Protein kinase (PKA) inhibitors, H-89 (Calbiochem International, San Diego, CA), U0126 and Bu2cAMP (Sigma, St Louis, MO) were also used in this study.
Plasmid constructs
Various lengths of human IGFBP-1 promoter-reporter constructs and CRE mutant construct were prepared previously (Gao et al., 1994; Gao et al., 1995; Gao et al., 1995) and are shown in Table I. Two sets of PRL promoter fragments (–2934 to –1404 bp) and (–1504 to +69 bp) were generated from HeLa cell DNA extracts using primers based on human decidual PRL promoter (Gellersen et al., 1994). The promoter fragments were inserted into pBL vector containing CAT reporter gene (Gao et al., 1994; Gao et al., 1995; Gao et al., 1995). Various lengths of PRL promoter-reporter constructs were prepared by restriction enzyme digestion as shown in Table I. Site-directed mutagenesis (Kunkel, 1985; Mazella et al., 2004) was carried out to locate the sequence(s) that mediate activation by RLX. Three mutants were constructed, pPRL322m [two C/EBP binding sites overlapping each other, –304 to –285 bp (Pohnke et al., 1999)], pPRL236m1 and pPRL236m2 [C/EBP binding sites in region, –214 to –201 bp and –81 to –66 bp, respectively] (Table I).
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Transfection assay
Endometrial stromal or decidual cells (0.8 x 106 cells/60 mm dish) were transfected with 8 µg of IGFBP-1 or PRL promoter constructs and 1 µg of pRSV-Luc. In stromal cells, promoter constructs were co-transfected with RLX-receptor (LGR7) expression vector (provided by Dr Hsueh (Hsu et al., 2002
). To determine the effect of RLX, cells were incubated with porcine RLX (20 ng/ml) for 48 h. Cells were then harvested in lysate buffer to determine the promoter activity. Promoter activity (CAT activity) was normalized to the luciferase activity derived from pRSV-Luc. Transactivation experiments were confirmed in at least 2–3 separate specimens. Representative data from a single experiment are shown in Figures 1 and 4–6. In each experiment, data are presented as mean ± SD (n=3, triplicate transfections), Statistical analysis was performed by paired Student's t-test. A P value of <0.05 was considered significant.
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Electrophoretic mobility gel shift assay and Western blot analysis
Nuclear extracts were prepared from endometrial stromal or decidual cells (1 x 108 cells each) by the method of Shapiro et al. (1988)
. The extracts (3–5 mg protein/ml) were kept in liquid nitrogen until use. Oligonucleotides of the IGFBP-1 promoter (Figure 3) were end-labelled with [32P]-
-dCTP (Amersham Corp., Arlington Heights, IL) and purified on a 4% polyacrylamide gel. The conditions of the protein/DNA binding reaction were as described previously (Gao et al., 1994). To determine whether CREB or p-CREB (phospho-CREB), in nuclear extracts would bind to the CRE, 1–2 µl of antibody specific to total CREB or p-CREB (phosphorylated at Ser133, Cell Signaling Technology Inc., Beverly, MA) was added to the reaction mixture and incubated for 30 min on ice and then for 30 min at 30 °C. DNA–protein complexes were resolved on 4% polyacrylamide gel and processed for autoradiography.
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To determine the effect of RLX on the phosphorylation of CREB in decidual cells, the cells were incubated with 20 ng/ml RLX for 0.5, 1, 2 and 24 h. Nuclear extracts were prepared after 24 h of incubation. The cell lysates were denatured by SDS buffer, boiled, resolved on 7% SDS–PAGE and then blotted onto PVDF membrane. CREB and p-CREB were identified by antibodies to CREB or p-CREB and detected by enhanced chemiluminescence. The relative intensities of protein/DNA band were scanned on Bio-Rad Image analysis system with Molecular Analyst/PC software.
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Results |
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RLX and LGR7 enhance the promoter activity of IGFBP-1 gene in decidual and endometrial stromal cells
To determine the effect of RLX on the promoter activity of IGFBP-1 gene, decidual cells were transfected with various lengths of the IGFBP-1 promoter-reporter constructs and then incubated with or without RLX for 48 h. RLX activated the promoter activity (Figure 1A). The highest increase was derived from cells transfected with pBP275. The activity was significantly reduced in cells transfected with the mutant construct, pBP275CREm or pBP246 in which a CRE site was deleted. To test whether the activation depends on the RLX receptor, we used endometrial stromal cells in which the RLX receptor content is much less than in decidual cells (
1/8 in stromal cells) (Mazella et al., 2004). Transactivation derived from pBP275 construct was observed only in cells co-transfected with LGR7 expression vector. Empty vector (without LGR7 insertion) had little effect on the activity (Figure 1B). These data show that the RLX is active in decidual cells which are abundant in RLX receptor and inactive in stromal cells that can be reversed by co-transfection with LGR7 expression vector.
RLX increases the phosphorylation of CREB and binding to CRE site in the IGFBP-1 promoter in decidual cells
To investigate whether the RLX receptor activates the cAMP dependent PKA system in decidual cells, we determined the time course of phosphorylation of the transcription factor CREB under the influence of RLX. Western blot analysis showed that p-CREB content in cell lysate was increased by RLX. The relative intensities of p-CREB at 0, 0.5, 1, 2 and 24 h were 1 (0 time), 8, 6, 4 and 3, respectively (Figure 2 upper panel), indicating that RLX stimulates phosphorylation of CREB. p-CREB content slightly decreased after 24 h incubation, but still remained above control levels in prolonged culture. After 24 h incubation with RLX, p-CREB content in the nuclear extracts was about 4-fold higher than that of control (Figure 2, last two lanes). The total CREB content remained similar, indicating that RLX does not alter the content of total CREB up to 24 h (Figure 2, lower panel). Results indicate that the RLX increased phosphorylation of CREB and p-CREB was accumulated in the nucleus.
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To characterize the binding patterns of CREB to the IGFBP-1 promoter, [32P] oligonucleotide containing the CRE site (Figure 3) of the IGFBP-1 promoter was used as the probe. Wild-type and mutant probes (radiolabelled to the same specific activity) were incubated with nuclear extracts of endometrial stromal or decidual cells (–RLX or + RLX for 24 h, Figure 3). There was little binding of the nuclear extracts to the [32P] mutant probe (mutation at CRE site, lane 1). Nuclear extracts from both stromal cells and decidual cells bound to the wild-type oligonucleotide forming two complexes, C1 and C2 [Figure 3, lanes 3, 4 [+ preimmune immunoglobulin G (IgG)], 7 and 10]. Addition of 10x cold CRE oligonucleotide reduced the intensity of C1 and C2 (lane 3 versus 2) indicating that binding to the CRE site is specific. To determine whether transcription factor, CREB, binds to the CRE site, antibody to total CREB was incubated with the reaction mixtures. The intensity of the C2 complex was reduced while there was increased intensity of the C1 complex (lanes 3, 4 versus 5; 7 versus 8, 11) suggesting that the C2 complex was formed by either CREB or p-CREB and it was supershifted by CREB antibody to the region of C1. When antibody to p-CREB was used, no effect on the C1 or C2 complexes formed by stromal cell nuclear extract was seen (lane 3, 4 versus 6), suggesting that the stromal is contain very little p-CREB. In contrast, p-CREB antibody reduced the intensity of both C1 and C2 formed by decidual cell nuclear extract binding (lanes 7 versus 9 and 10 versus 12). This indicates that C1 and C2 were at least partially formed by p-CREB. Nuclear extracts from RLX treated decidual cells increased binding to the CRE site (3-fold increase, lane 7 versus 10). The increase of p-CREB binding correlated with the RLX-enhanced transactivation (Figure 1).
RLX and LGR7 enhance the promoter activities of PRL in decidual and endometrial stromal cells
The effect of RLX or RLX/LGR7 on PRL gene promoter activity was determined in decidual and stromal cells. The pPRL3.0 and pPRL1.6 (3 and 1.6 kb) promoter constructs were activated by RLX (data not shown). Figure 4 shows the activities derived from constructs with the shorter promoter constructs, –401, –322, –236 and –59 to +69 bp and pPRL322 mutant. The highest activation was observed in cells transfected with pPRL322 (Figure 4). Transactivation induced by RLX was significantly reduced when cells were transfected with a mutant construct, pPRL322m (mutation of the two C/EBP binding sites, –304 to –297 bp and –298 to –285 bp) or constructs with deletion of C/EBP binding sites, pPRL236 or pPRL59 (Figure 4A). Mutant constructs, pPRL236m1 and pPRL236m2 (Table I), had similar activity compared to that of wild-type construct, pPRL 236 (data not shown). Figure 4B shows the profiles of the promoter activities in endometrial stromal cells. Little activation was observed when cells were co-transfected with empty vector. RLX significantly increased the activities derived from pPRL401 and pPRL322 when co-transfected with LGR7 expression vector (Figure 4B). Activity was significantly reduced in cells transfected with pPRL322m and LGR7. The active sites mediated by RLX (Figure 4) coincided with the sites that mediate the activation by Br2cAMP (Pohnke et al., 1999). These data suggest that the effect of RLX on the PRL promoter activity is also mediated by cAMP activated signalling pathway.
Effect of PKA and MAPK inhibitors, H-89 and U0126, on RLX enhanced IGFBP-1 and PRL promoter activities
RLX is capable of activating both PKA and MAPK (Zhang et al., 2002). To provide additional evidence as to whether RLX enhanced-activation also involves MAPK, we used the PKA and MEK1/MEK2 (mitogen-activated protein kinase kinase) specific inhibitors H-89 and U0126, respectively (Chijiwa et al., 1990; Favata et al., 1998), to determine their effect on IGFBP-1 and PRL promoter activity. Figure 5A shows that RLX enhanced IGFBP-1 promoter activity was significantly inhibited by H-89, but it was not affected by U0126. In contrast, PRL promoter activity was significantly inhibited by both H-89 and U0126. These results indicate that RLX mediated target gene activation may involve PKA alone or PKA and MAPK depending on which transcription factor(s) and cis-element(s) are involved in endometrial cells.
Relative capacity of RLX and Bu2cAMP on the activation of IGFBP-1 and PRL promoters
The activation of IGFBP-1 and PRL gene transcription by various doses of RLX or Bu2cAMP were investigated to compare the capacity of endogenous RLX receptor and PKA in decidual cells. Figure 6A and B shows that the induction capacity was similar in cells treated with 2, 20 or 100 ng/ml RLX indicating that endogenous RLX receptor level may be saturated which limits its capacity for additional activation. A pharmacological dose of Bu2cAMP (0.05 mM) was required to increase the promoter activity by 2–3-fold, a little less than that derived from 2 ng/ml RLX. Activation by 0.5 and 1 mM Bu2cAMP were 2–3-fold higher than that of RLX, suggesting that endometrial PKA(s) has higher capacity than RLX receptor for cAMP mediated gene activation.
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Discussion |
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Activation of PKA by RLX in target tissues has been examined in various types of mammalian cells as well as in endometrial glandular and stromal cells (Chen et al., 1988
; Tseng et al., 1999; Hsu et al., 2002; Zhang et al., 2002; Bartsch et al., 2004). The current study presented evidence that the RLX-induced IGFBP-1 and PRL gene activation is mediated through a CRE site and C/EBP binding sites in the IGFBP-1 and PRL promoters, respectively.
We have shown that RLX enhanced activation requires co-transfection of the LGR7 expression vector in stromal cells, whereas in decidual cells RLX directly activates the promoter activities. Results indicate that lower levels of RLX receptor content in stromal cells (1–5 copies/cells) (Mazella et al., 2004) may be insufficient to sustain cAMP accumulation due to high activity of PDE (Bartsch et al., 2004). Presumably, higher levels of RLX receptor increases the rate of cAMP production to counteract the PDE activity, and therefore enhance PKA activity for the phosphorylation of CREB (Figure 2).
Zhang et al. (2002) have shown that the RLX induces phosphorylation of CREB, MAPK and MEK, which peaks at 10 min of incubation and it reduced to basal levels at 30 min in endometrial stromal cells. In decidual cells, maximal phosphorylation was maintained at 0.5 h and remained above control levels over the 24 h incubation period (Figure 2) suggesting that the RLX is able to enhance phosphorylation of CREB in both types of cells, but PKA activity is stronger in decidual cells due to higher RLX receptor levels. Thus, it sustains the cAMP mediated activities. Alternatively, one could interpret these observations as endometrial PDE activities. PDE4 inhibitor is able to enhance the RLX-induced IGFBP-1 and PRL production (Bartsch et al., 2004) indicating that PDE is a key factor that controls the RLX enhanced PKA activity in stromal cells. PDE activity may also be active in decidual cells since PDE is up-regulated by cAMP analogue (Oki et al., 2000). However, cellular p-CREB was retained much longer in decidual cells (Figure 2) than in stromal cells (Zhang et al., 2002). This suggests that the steady state between phosphorylation and dephosphorylation is shifted in favour of cAMP accumulation resulting in a higher level of nuclear p-CREB, more binding to the CRE site (Figure 3) and transcriptional activity of IGFBP-1 (Figure 1). Further investigations are necessary to establish the different mechanisms involved.
Figure 2 also shows that elevated p-CREB was maintained in the nucleus in the presence of RLX after 24 h. It has been shown that forskolin or ethanol stimulates the translocation of the catalytic subunit of type II PKA into the nucleus and enhances the phosphorylation of CREB (Constantinescu et al., 2004). Although we showed that p-CREB was accumulated in the nucleus (Figure 2), whether or not that RLX enhances nuclear translocation of the catalytic subunit of PKA is unknown.
H-89 PKA inhibitor, but not U0126, abolished RLX-induced IGFBP-1 promoter activity (Figure 5). In addition, RLX increased the binding capacity of decidual nuclear extract to the CRE site. Data indicate that transcriptional activation of IGFBP-1 is mediated by PKA activation alone. In contrast, both H-89 and U0126 inhibited RLX-induced PRL promoter activity (Figure 5) suggesting that the C/EBP activation involves both PKA and MAPK signalling pathways. Data imply that multi-signalling pathways are involved in C/EBP induced gene activation.
Induction of IGFBP-1 and PRL correlated with increasing concentrations of Bu2cAMP, whereas induction by RLX was saturated at 2 ng/ml of RLX (Figure 6). These data suggest that RLX receptor or RLX receptor coupled G-protein(s) is the rate limiting factor that controls the extent of gene activation. Alternatively, RLX content may be quickly degraded to prevent a dose-dependent response in the culture system.
Despite limited capacity of RLX receptor, the effect of RLX is physiologically important. Previous studies have shown that RLX and progestin increase IGFBP-1 production in stromal cells and RLX alone increases IGFBP-1 production in decidual cells with an overall increase that exceeds 8000-fold (Mazella et al., 2004). Decidual PRL is also increased similarly (Zhu et al., 1990; Bell et al., 1991; Tseng et al., 1992; Lane et al., 1994; Mazella et al., 2004). Thus, RLX mediated activation may be one of the major signalling pathways that occurs in stromal/decidual cells during gestation. Of course, other peptide hormones, such as HCG also should be considered.
In summary, we have shown that the RLX induces IGFBP-1 and PRL transcription in decidual cells and LGR7 enhances the RLX-induced transcription in stromal cells. These results relate with high production rates of IGFBP-1 and PRL in stromal and decidual cells and they mimic the high levels of IGFBP-1 and PRL accumulated in amniotic fluid during gestation (Rosenberg et al., 1980; Bell et al., 1986). Therefore, RLX receptor induced PKA activity is physiologically important for activation of decidual cell IGFBP-1 and PRL.
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Acknowledgements |
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We thank Dr A.Hsueh for providing us the LGR7 expression vector. We also thank clinicians in the Department of Obstetrics/Gynecology and Reproductive Medicine and Department of Pathology and the Labor and Delivery division at SUNY-Stony Brook for providing us with viable endometrial specimens. This article is supported by NIH grant HD 19247.
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Submitted on December 16, 2004; accepted on January 4, 2005.
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J. Mazella, S. Liang, and L. Tseng Expression of Delta-Like Protein 4 in the Human Endometrium Endocrinology, January 1, 2008; 149(1): 15 - 19. [Abstract] [Full Text] [PDF]
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 O. Eyal, J.-B. Jomain, C. Kessler, V. Goffin, and S. Handwerger Autocrine Prolactin Inhibits Human Uterine Decidualization: A Novel Role for Prolactin Biol Reprod, May 1, 2007; 76(5): 777 - 783. [Abstract] [Full Text] [PDF]
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 W. Zhang, J. Mazella, H. J. Kloosterboer, and L. Tseng Progestagenic Effects of Tibolone are Target Gene--Specific In Human Endometrial Cells Reproductive Sciences, September 1, 2006; 13(6): 459 - 465. [Abstract] [PDF]
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 J. L. Sarno, F. Schatz, C. J. Lockwood, S.-T. J. Huang, and H. S. Taylor Thrombin and Interleukin-1{beta} Regulate HOXA10 Expression in Human Term Decidual Cells: Implications for Preterm Labor J. Clin. Endocrinol. Metab., June 1, 2006; 91(6): 2366 - 2372. [Abstract] [Full Text] [PDF]
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