Department of Obstetrics and Gynecology, School of Medicine, State University of New York at Stony Brook, Stony Brook, NY 11794-8091, USA
1 To whom correspondence should be addressed. e-mail: litseng{at}notes.cc.sunysb.edu
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Abstract |
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Key words:
endometrial decidual and stromal cells/relaxin/relaxin receptor/TGF/IGFBP-1
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Introduction |
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Among various types of factors, relaxin (RLX) and transforming growth factor (TGF) have been considered to be the essential factors for tissue remodelling in endometrium (Lane et al., 1994
; Tseng, 1994
; Parekh et al., 2002
). It has been shown that these two factors are expressed in endometrial tissue (Casslen et al., 1998
; Palejwala et al., 2002
; Parekh et al., 2002
). However, the target genes activated by RLX or TGF
appear to be different in endometrial cells.
The biological effect of RLX on endometrium was first demonstrated four decades ago by Hisaw and Hisaw (1964), who showed that administration of RLX in conjunction with estrogen and progesterone resulted in a pregnant-like endometrium in monkey. More recent studies from our laboratory have shown that RLX enhances decidualization based on the ultrastructural analysis of human endometrial stromal cells in long-term culture under the influence of medroxyprogesterone acetate (MPA) and RLX (Lane et al., 1994
). RLX causes a rapid increase in cAMP accumulation and induces glycodelin production in human endometrial glandular epithelial cells (Chen et al., 1988
; Tseng et al., 1999
). In endometrial stromal cells, accumulation of cAMP and target gene activation were noticed after a long period of incubation with progestin and RLX (Tseng et al., 1992
; Tseng, 1994
). It is unclear how progestin enhances the RLX action or vice versa. We hypothesized that progestin increases RLX receptor content which determines the function of RLX.
Although binding of RLX to its receptor has been demonstrated in numerous publications, RLX receptor protein was recently cloned and identified as two orphan leucine-rich G-protein-coupled receptors, LGR7 and LGR8 (Hsu et al., 2002; Hsu, 2003
). Subsequently, RLX receptor mRNA and protein expression in human and primate endometrium have been identified in both the epithelial and stromal compartments (Ivell et al., 2003
; Luna et al., 2004
). Hormone, however, appears to be ineffective in regulating LGR7 mRNA (Ivell et al., 2003
). In this study we investigated the effect of hormones on RLX receptor in endometrial cells by real-time PCR which is able to quantify low levels of mRNA and to detect any changes on the level of message affected by the hormone. In addition to endometrial stromal and glandular cells, decidual cells were included in this study.
TGF has been claimed to play a role in endometrial cell differentiation. It has been shown to enhance tissue remodelling and homeostasis in endometrial cells (Bruner et al., 1995
; Casslen et al., 1998
; Ulloa and Tabibzadeh, 2001
) and inactivation of TGF
has been implicated to endometrial carcinogenesis (Parekh et al., 2002
). However, it is unclear whether TGF
would regulate those proteins that are abundantly produced in decidual cells, such as IGFBP-1 and PRL. In this study, we have examined the effect of RLX and TGF
to determine whether these two factors have similar or disparate effects on RLX receptor and the induction of the decidual marker protein, IGFBP-1.
Although it is well known that decidual cells secrete a large amount of IGFBP-1, regulation by hormones in decidual cells is unclear. We have investigated the induction patterns of IGBP-1 in decidual and stromal cells, run in parallel.
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Materials and methods |
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Stromal and glandular epithelial cells (>95% purity) were isolated from endometrium by digesting the tissue fragments with collagenase as described previously (Zhu et al., 1990; Lane et al., 1994
). Decidual layer attached to the fetal membrane contains a mixture of decidual fibroblasts, newly formed decidual cells, and terminally differentiated decidual cells. Decidual cells were isolated by a method described previously (Tseng, 2003
). Briefly, the decidual layer was dissected from the fetal membrane, washed extensively with saline, minced into small pieces and digested with 0.25 mg/ml collagenase P (Roche, USA) for 2030 min to loosen the tissue structure. The remaining tissue fragments were dispersed into cell suspension by 0.05% trypsin for 5 min. Cells were subcultured twice to eliminate the non-adherent cells (which includes terminally differentiated decidual cells, vascular endothelial cells and blood cells). Newly formed decidual cells are able to attach to the surface of a Petri dish. Decidual cells (
95% purity) were identified by positive immunohistochemical staining of 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 individually cultured (no mixed culture from different specimens). To study the effect of hormone, culture medium RPMI (Roswell Park Memorial Institute 1640 medium and 2% charcoaldextran-stripped fetal bovine serum) was supplemented with progestin, MPA, porcine RLX (Sherwood et al., 1993
) and human recombinant TGF
1 (Sigma, USA), separately or in combination for various periods of time specified in each experiment.
Determination of LGR7 and LGR8 mRNA by real-time PCR
Total RNA was isolated from cells using RNAqueous Kit (Ambion, USA). We used real-time PCR for quantification of LGR7 and LGR8 mRNA. Real-time PCR quantifies the message at the exponential growth phase of PCR product (Figure 1). Optimal primers used in this study are shown in Table I. Primers of LGR7(1) were selected from reported cDNA (Hsu et al., 2000) to detect the full length of LGR7 mRNA only and primers of LGR7(1,2) were designed to detect both the full length and a possible splicing variant, missing 34 amino acids (Hsu et al., 2000
). The two messages can be distinguished by the length of their PCR products, 204 bp for full length and 102 bp for the splicing variant. QuantiTec SYBR Green PCR Kit (204143; Qiagen, USA) was used for real-time PCR to quantify the mRNA. Briefly, 5 µg total RNA was reversed-transcribed (RT) using random primers. Diluted RT products (equivalent 0.25 µg total RNA) were subject to real-time PCR [10 µl SYBR Green Master mix, 1 µl (8 pmol/µl) each forward and reverse primer and 8 µl cDNA]. Standard curves were generated from either plasmids containing the gene of interest or from amplified PCR product.
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Fibronectin synthesized and secreted from decidual cells determined by [35S]methionine labelling
Fibronectin (FN) synthesis was determined by [35S]methionine labelling and immunoprecipitation (Zhu et al., 1990). Cells were incubated with 0.1 mCi/ml [35S]methionine/cysteine (SA >1000 Ci/mmol; NEN Life Science, USA) in methionine free culture media for 3 h. Labelled medium (0.5 ml: one-third of total volume) was incubated with Mab of FN (ICN Biochemicals, USA) and absorbed to protein G agarose beads. After extensive washing, the agarose beads were boiled in the sodium dodecyl sulphate (SDS) loading buffer and analysed by SDSpolyacrylamide gel electrophoresis (PAGE). Authentic FN was run in parallel.
Statistical analyses
LGR7 mRNA content in endometrial stromal, glandular and decidual cells, and induction of LGR7 mRNA by progestin or RLX, were performed in number of specimens specified in each condition. For P-value computation, statistics was performed by Students unpaired t-test on the levels of LGR7 mRNA in stromal, glandular and decidual cells and paired t-test on the induction by hormones. P < 0.05 was considered significant.
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Results |
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Discussion |
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The effect of progestin on the induction LGR7 mRNA is likely caused by progestin-induced stromal cell differentiation since the process of induction is slow and it requires 5 days incubation for a noticeable increase of LGR7 mRNA. Also, the extent of induction in stromal cells is relatively small compared to that in decidual cells. These observations suggest that other unidentified cellular factors are also involved. The up-regulation of LGR7 mRNA by RLX, however, is time- and dose-dependent and it is likely that RLX exerts a direct effect on the transcription of LGR7 mRNA.
In this study, we presented a function of LGR7 (induction of IGFBP-1) rather than detecting the receptor protein in endometrial cells. The extent of induction of IGFBP-1 is proportional to the amount of LGR7 mRNA, i.e. a small induction correlates with the low level of LGR7 mRNA in stromal cells and a large induction correlates with the higher mRNA content in decidual cells. Thus the mRNA content reflects the relative content of LGR7 protein in endometrial cells.
It should be mentioned that other factors, namely, prostaglandin (PGE2), hCG, FSH or LH and cAMP generating agents exhibit similar or redundant functions as RLX to enhance the production of IGFBP-1 and PRL (Tang and Gurpide, 1993; Frank et al., 1994
; Telgmann et al., 1997
). The effect of RLX appears to be more effective than the others. As shown in the present study, the induction of LGR7 is highly specific. In addition, the stimulation of IGFBP-1 is sustained from sub-microgram in stromal cells to microgram levels in decidual cells with an overall increase >10 000-fold (Figure 5A and B). Maximal production is similar to the accumulation of IGFBP-1 in amniotic fluid during early to mid gestation (Bell et al., 1988
). Similar results were observed on the production of PRL in endometrial cells, i.e. synergistic effect induced by MPA/RLX in stromal cells, major induction by RLX in decidual cells and inhibition by TGF
1 in both types of endometrial cells (data not shown). These results imply that the productions of endometrial IGFBP-1 and PRL are sustained by RLX in decidual cells during gestation.
As shown in Figures 3 and 5, TGF1 had an inhibitory effect on the content of RLX receptor and IGFBP-1 production. The degrees of inhibition by TGF
1 shown in Figure 3 are different in two types of endometrial cells. In general, inhibition by TGF
1 is greater in stromal cells than in decidual cells, suggesting that decidual cells may contain factor(s) that neutralize the effect of TGF
1. One such factor could be lefty, which is known to be highly expressed in deciduas (Brar et al., 2001
). Further study is required to elucidate the roles of these cytokines in deciduas.
The inhibitory effect of TGF was unexpected since it is believed that TGF
promotes cell differentiation of numerous mammalian systems by stimulating the production of extracellular matrix proteins, e.g. induction of fibronectin in mammalian cells (Hocevar et al., 1999
) and in decidual cells (Figure 4). However, endometrial cells cultured with TGF
had no noticeable change in morphology (not shown). On the other hand, stromal cells treated with MPA and RLX exhibited changes in morphology associated with decidualization (Lane et al., 1994
). The inhibitory effect on LGR7 and IGFBP-1 by TGF
1 suggests that it has little contribution to endometrial cell decidualization. Further study is necessary to elucidate the inhibitory effect of TGF
on RLX-mediated signalling transduction pathways.
In summary, we showed that TGF1 inhibits the induction of LGR7 mRNA and IGFBP-1. Sequential stimulation by progestin and then RLX is the major driving force for the production of IGFBP-1. RLX exerts a direct effect to induce LGR7 and IGFBP-1 production in decidual cells. Thus, we conclude that RLX receptor is a rate-limiting factor for the biological functions of RLX and it is essential to sustain the production of decidual cell marker protein.
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Acknowledgements |
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References |
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Submitted on January 27, 2004; accepted on March 24, 2004.