Molecular Human Reproduction, Vol. 8, No. 11, 992-997,
November 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Regulation of follistatin-related gene (FLRG) expression by protein kinase C and prostaglandin E2 in cultured granulosa-luteal cells
1 Department of Pathology, P.O. Box 21, University of Helsinki, FIN-00014 Helsinki, 2 Department of Paediatrics, P.O. Box 1777, Kuopio University and University Hospital, FIN-70211 Kuopio, and 3 Department of Obstetrics and Gynaecology, P.O. Box 140, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland
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Abstract |
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Activin and its binding protein follistatin may act as local regulators of cell growth and steroidogenesis in the human ovary. The recently identified follistatin-related gene (FLRG) is expressed abundantly in the human ovary, has high affinity for activin, and is able to inhibit activin-induced transcriptional responses. However, little is known about the regulation of FLRG expression in specific cell types in the ovary, while it is known that gonadotrophins induce follistatin gene expression in human granulosa-luteal cells. In this study, we investigated the expression of FLRG mRNA in granulosa-luteal cells of preovulatory follicles obtained from women undergoing IVF. FLRG mRNA was detected by RT–PCR in fresh and cultured granulosa-luteal cells, as well as in normal ovarian stroma, theca and granulosa cells. Northern blot analysis revealed a 2.5 kb transcript of the FLRG in cultured granulosa-luteal cells. The protein kinase C activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 160 nmol/l), and prostaglandin E2 (PGE2, 1 µmol/l) increased FLRG mRNA accumulation up to 3–8 fold over the control level after 24 h of treatment, and these stimulatory effects were dose-dependent. Co-treatment with the protein kinase C inhibitor, Ro-31–8220 (3 µmol/l), blocked the stimulatory effect of TPA. Although short term treatment with the protein kinase A activator, (Bu)2cAMP (1 mmol/l), slightly reduced FLRG mRNA expression in most experiments, long term treatment with FSH (100 IU/l), LH (100 IU/l), or (Bu)2cAMP had no significant effect on the FLRG mRNA levels. As expected, gonadotrophins, protein kinase A and C activators and PGE2 increased granulosa-luteal cell progesterone secretion into the culture media. Taken together, previous and our present data suggest that protein kinase C and A signal transduction pathways differently regulate the expression of FLRG and follistatin genes in human ovarian granulosa-luteal cells.
follistatin-related gene/gonadotrophin/granulosa-luteal cells/protein kinase A/protein kinase C
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Introduction |
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Inhibins and activins belong to the transforming growth factor-ß (TGF-ß) superfamily. They are structurally related dimeric glycoprotein hormones initially characterized by their ability to suppress and stimulate, respectively, FSH secretion by the pituitary gland (Ying, 1988
; Lockwood et al., 1998). Recent studies have indicated that activins may also act as important local regulators of cell growth and steroidogenesis in the ovary (Peng et al., 1996; Findlay et al., 2000). Activin-A suppresses progesterone secretion and cholesterol side-chain cleavage enzyme (P450scc) gene expression, but stimulates cellular proliferation in human granulosa-luteal cell cultures (Rabinovici et al., 1990; Erämaa et al., 1995; Peng et al., 1996; Alak et al., 1998). The biological effects of activins are regulated through multiple mechanisms. Follistatin, an intensively studied activin binding protein, is a single chain glycoprotein originally isolated from bovine and porcine ovarian follicular fluid as a substance suppressing FSH secretion. After initial purification, follistatin was found to bind both activin and inhibin with high affinity through their common ß-subunits. Follistatin prevents the access of activins to their cellular receptors, and thus neutralizes their biological effects (Michel et al., 1993; Phillips and de Kretser, 1998). As in the case of the inhibin/activin subunits, the follistatin gene is also expressed in human granulosa cells. In cultured granulosa-luteal cells, follistatin expression is regulated by gonadotrophins through both protein kinase A and C dependent pathways, and exogenous follistatin antagonizes the effects of activin-A on steroidogenesis (Cataldo et al., 1994; Tuuri et al., 1996).
Follistatin is a member of a large group of proteins containing a highly conserved module of cysteine-rich sequence termed the follistatin domain. This family includes follistatin, follistatin-related gene (FLRG) protein, follistatin-related protein (FRP), agrin, secreted protein acidic and rich in cysteine (SPARC), and Mac25 (Hayette et al., 1998; Phillips and de Kretser, 1998). However, most proteins in this family have no follistatin-like activity in respect of regulating the biological function of activins. Besides follistatin, in this group only FLRG protein has been reported to bind TGF-ß superfamily members (Tsuchida et al., 2000; Tortoriello et al., 2001). FLRG was originally cloned in the molecular study of a t(11;19)(q13;p13) translocation observed in a case of B-cell chronic lymphocytic leukaemia (Hayette et al., 1998). Like follistatin, FLRG protein binds activin with high affinity and neutralizes its biological activity (Tsuchida et al., 2000; Tortoriello et al., 2001). FLRG is expressed in a wide range of human tissues including steroidogenic glands such as adrenals, testes and ovaries (Hayette et al., 1998; Tsuchida et al., 2000; Tortoriello et al., 2001), indicating that FLRG may also function through autocrine/paracrine pathways to affect the activity of activins in these tissues. Although TGF-ß and activin A have been shown to increase the transcription of the FLRG gene through Smad proteins in the human hepatoma HepG2 cell line and bone marrow stromal cells (Bartholin et al., 2001; Maguer-Satta et al., 2001; Bartholin et al., 2002), little is known about the regulation of FLRG expression in specific cell types in the ovary. Since inhibin/activin subunit and follistatin gene expression is co-ordinately regulated by gonadotrophins in human granulosa-luteal cells (Peng et al., 1996), we hypothesized that FLRG may also be expressed and regulated hormonally in these cells. To clarify the regulation of FLRG expression in ovaries, we studied the effects of gonadotrophins and protein kinase modulators on the FLRG mRNA levels in cultured human granulosa-luteal cells.
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Materials and methods |
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Ethical considerations
The study protocols were approved by the local research ethics committees (Voutilainen et al., 1996
; Liu et al., 2001). The patients gave informed written consent.
Ovarian RNA samples
Ovarian RNA originated from previously described women with regular cycles (Voutilainen et al., 1996). The ovaries were collected during the follicular phase. The women had not received any medication for stimulation or suppression of ovulation for at least 3 months prior to surgery.
Cell cultures
Human granulosa-luteal cells were obtained by follicular aspiration from women undergoing oocyte retrieval for IVF. The cells were either used directly for extraction of total RNA, or cultured and treated as previously described (Liu et al., 2001). In order to allow the cells to recover the responsiveness to gonadotrophins, they were cultured for 5–10 days in medium containing fetal calf serum before initiating the treatments (Schipper et al., 1993). Our previous studies have demonstrated that at this culture stage, human granulosa-luteal cell progesterone production and inhibin/activin subunit mRNA expression are optimally responsive to gonadotrophin treatment (Voutilainen et al., 1986; Vänttinen et al., 2000; Liu et al., 2001). The cell culture experiments were carried out in triplicate dishes and each experiment was repeated at least three times to make sure that the results were reproducible.
Recombinant human FSH (rhFSH) and LH (rhLH) used for cell culture experiments were gifts from Serono-Nordic (Vantaa, Finland). (Bu)2cAMP, 12-O-tetradecanoyl phorbol 13-acetate (TPA), prostaglandin E2 (PGE2) and F2
(PGF2) were purchased from Sigma (St Louis, MO, USA), and Ro-31–8220 was from Calbiochem (Darmstadt, Germany).
RNA extraction and Northern blot analysis
Total RNA was previously isolated from different ovarian compartments (Voutilainen et al., 1996) and now from fresh granulosa-luteal cells (IVF patients) by ultracentrifugation through a cesium chloride cushion (Chirgwin et al., 1979). Cytoplasmic RNA was extracted from the cultured cells based on a previously described method (Voutilainen et al., 1986). Northern blotting and hybridizations were performed as described previously (Liu et al., 1997). The FLRG mRNA was detected with a synthetic oligonucleotide complementary to human FLRG mRNA. The sequence of this oligonucleotide was 5'-GAGATGTAGGTGACGTTGTTGTTGCCGCAA-3', corresponding to the nucleotides 637–666 of the FLRG mRNA (GenBank Accession no. U76702) (Hayette et al., 1998). The oligonucleotide and mouse ribosomal 28S RNA cDNA used as a loading control (Arnheim, 1979) were labelled as described previously (Liu et al., 1997). The relative intensities of the autoradiographic signals were quantified by densitometric scanning. All RNA data shown here were normalized with the respective 28S ribosomal RNA values.
RT–PCR
Due to the lack of availability of normal ovarian tissues, RT–PCR analysis of previously isolated RNA (Voutilainen et al., 1996) was used to investigate FLRG expression in different ovarian compartments. The PCR was performed with denaturation at 95°C for 5min; 40 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 30s; and extension at 72°C for 10min. The primer set was 5'-GGCAACAACAACGTCACCTA-3' (sense, nucleotides 641–660 of GenBank Accession no. U76702) and 5'-CCTAAATCAGGCGCTCAGTG-3' (antisense, nucleotides 1016–1035). The above mentioned FLRG specific oligonucleotide for Northern blot analysis was used for verification of the specificity of the PCR reaction products. Normal human adrenal RNA (Liu et al., 1997) was used as a positive control.
Progesterone measurement
Progesterone was measured by a competitive enzyme immunoassay (EIA) purchased from Diagnostic Systems Laboratories, Inc. (Webster, TX, USA). The detection limit of the assay was considered 1 nmol/l. The intra- and interassay coefficients of variation were 7.5 and 9.4% respectively.
Statistical analyses
The differences in the FLRG mRNA levels (based on densitometric measurements and corrected for loading in Northern blots) and progesterone concentrations were assessed by the non-parametric Mann–Whitney test. The level of significance was chosen as P < 0.05.
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FLRG mRNA was detectable in normal whole ovaries as well as in all ovarian compartments (granulosa, theca and stroma cells) by RT–PCR. The granulosa-luteal cells freshly collected during IVF or cultured for 8 days without gonadotrophin stimulation also expressed FLRG mRNA. As a positive control, FLRG mRNA was readily detectable in normal human adrenals (Figure 1
).
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In our cultured granulosa-luteal cells, both inhibin A and B secretion is detectable (Vänttinen et al., 2000
), and all three inhibin/activin subunit mRNAs are expressed (Liu et al., 2001; and data not shown). FLRG mRNA was detectable by Northern blotting in all cell experiments after 1–10 days of culture. As shown in Figure 2, a 2.5 kb transcript of the FLRG was hybridized with our oligonucleotide probe in granulosa-luteal cells maintained in culture for 8 days.
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Since gonadotrophins and protein kinase A activator cAMP up-regulate inhibin/activin
and ßA subunits as well as follistatin mRNA expression in granulosa-luteal cells (Tuuri et al., 1996), we tested whether the steady state mRNA levels of the FLRG are also regulated by these agents. Neither rhFSH nor rhLH (both at the concentration of 100 IU/l) affected the FLRG mRNA accumulation significantly during short (3 h) or long term (24–48 h) treatments (data not shown; all P > 0.05, n = 4 experiments for each analysis). Consistently, activation of protein kinase A by (Bu)2cAMP also did not have any effect on the FLRG mRNA accumulation after 24 h of treatment (Figure 2
). However, in short term (3 h) treatment, (Bu)2cAMP (1mmol/l) slightly reduced (maximally 25%) FLRG mRNA accumulation (P < 0.05, n = 4) (data not shown).
For clarifying the effect of the protein kinase C pathway on FLRG mRNA expression in granulosa-luteal cells, we treated the cells with the protein kinase C activator TPA and inhibitor Ro-31–8220. Interestingly, the FLRG mRNA accumulation was significantly increased by TPA treatment after 24 h of incubation (Figure 2). During short term (3 h) treatment, TPA (160 nmol/l) tended to increase FLRG mRNA expression slightly but this was not significant on the basis of three separate experiments (P = 0.12). The stimulatory effect of TPA was dose-dependent during long term treatment, from the concentration of 16 nmol/l upwards, reaching maximal stimulation (up to 3–8 fold over the control level) at 160 nmol/l (Figure 3). Although the protein kinase C inhibitor Ro-31–8220 (3 µmol/l) alone had no influence, it totally blocked the stimulatory effect of protein kinase C activator TPA (160 nmol/l) on FLRG mRNA accumulation after 24 h of co-treatment (Figure 2).
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For identifying possible natural ligands regulating FLRG expression, we performed experiments with prostaglandins which have previously been reported to activate protein kinase C (Abayasekara et al., 1993
; Narumiya et al., 1999). Our results demonstrated that PGE2 upregulates FLRG expression in cultured granulosa-luteal cells after 24 h of treatment. This stimulatory effect of PGE2 was dose-dependent (Figure 4). No significant change was detected after short term (3 h) incubation with PGE2. In contrast, PGF2 had no significant effect on FLRG mRNA accumulation (data not shown).
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Although gonadotrophins and (Bu)2cAMP did not increase FLRG mRNA expression in long term treatments, these cells did respond to these agents as they increased progesterone secretion into the culture media in both long and short term experiments. As shown in Figure 5
, progesterone secretion was increased up to 357 ± 74% (mean ± SE) of the control (n = 16 experiments, P = 0.0001) by rhFSH (100 IU/l), up to 331 ± 102% (n = 10, P = 0.0001) by rhLH (100 IU/l), and up to 735 ± 128% (n = 12, P = 0.0001) by (Bu)2cAMP (1 mmol/l) in long term treatments. In similar experiments FSH, LH and (Bu)2cAMP also increased inhibin A and B secretion (Vänttinen et al., 2000). Consistent with a previous report (Hori et al., 1998), long term treatment with TPA also increased progesterone secretion up to 252 ± 59% of the control (n = 11, P < 0.01) (Figure 5). Co-treatment with TPA attenuated the stimulatory effect of (Bu)2cAMP on progesterone secretion by 35%, but the secretion remained higher than the basal progesterone secretion (Figure 5). In addition, PGE2 increased progesterone secretion dose-dependently up to 344 ± 33% of the control (n = 6, P < 0.005) at the concentration of 1 µmol/l after 24 h of treatment (Figure 5) (dose response data not shown).
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Discussion |
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Our Northern blot and RT–PCR analysis demonstrated that FLRG is expressed in human ovarian granulosa and granulosa-luteal cells, as well as in theca and stroma cells, confirming the previous report of FLRG expression in the human ovary (Hayette et al., 1998
; Tortoriello et al., 2001). We found that FLRG mRNA accumulation is up-regulated by the protein kinase C activator TPA, and the stimulatory effect of TPA is blocked by the protein kinase C inhibitor Ro-31–8220. This is in agreement with a recent report that mouse FLRG promoter activity is augmented by TPA treatment (Nakatani et al., 2002). In contrast, activation of protein kinase A by (Bu)2cAMP, FSH and LH had no significant effect on the FLRG mRNA expression, although in short term experiments (Bu)2cAMP tended to decrease FLRG mRNA accumulation. The physiological significance of the stimulatory effect of protein kinase C activation on the FLRG expression in granulosa-luteal cells remains to be clarified.
It has previously been reported that PGF2, gonadotrophin-releasing hormone and adenosine triphosphate regulate human granulosa-luteal cell functions through the protein kinase C pathway (Abayasekara et al., 1993; Kang et al., 2001; Tai et al., 2001). Interestingly, we found that PGE2, instead of PGF2, increases the expression of FLRG mRNA in our culture model. PGE2 is known to be produced in corpus luteum and to exert its effect through binding to cell surface EP receptors (Olofsson and Leung, 1994). Human granulosa-luteal cells express functional EP1 and EP2 prostaglandin receptors (Harris et al., 2001), both of which bind PGE2 better than PGF2. The activation of EP1 and EP2 receptors is associated with an increase in intracellular Ca2+ or cAMP concentrations respectively (Narumiya et al., 1999). The ability of EP1 and EP2 receptor activation to induce distinct second messenger responses may allow PGE2 to induce different physiological actions depending on cohort activities of different protein kinases. Our data demonstrate that FLRG is up-regulated by PGE2 in granulosa-luteal cells. On the basis of the inability of (Bu)2cAMP to induce FLRG expression, it may be suggested that the effect of PGE2 on FLRG is transmitted through the EP1 receptor and an increase in intracellular Ca2+ concentration (Narumiya et al., 1999).
FLRG protein has high affinity for activin, and is able to inhibit activin-induced transcriptional responses (Tsuchida et al., 2000; Sidis et al., 2002). Therefore, the induction of FLRG expression by protein kinase C activation and PGE2 may be involved in the regulation of cellular signalling of activin in granulosa-luteal cells. It has previously been reported that activin A reduces steroidogenesis in human granulosa-luteal cells (Rabinovici et al., 1990; Erämaa et al., 1995; Alak et al., 1998). Although TPA and PGE2 can increase inhibin/activin ßA subunit mRNA expression (Tuuri et al., 1996) and activin A secretion (Miyanaga et al., 1993) in granulosa cells, they increased progesterone production in our study. This stimulation of steroidogenesis by TPA and PGE2 could at least partly be explained by TPA and PGE2 induced FLRG (this study) and follistatin (Tuuri and Ritvos, 1995; Tuuri et al., 1996) expression, being able to block the inhibitory effect of activins on progesterone secretion. However, the different time course of the stimulatory effect of TPA and PGE2 on follistatin and FLRG expression suggests different roles for these two binding proteins in the regulation of activin bioactivity in granulosa cells. The stimulatory effect of TPA and PGE2 on follistatin mRNA levels appears rapidly (Tuuri and Ritvos, 1995; Tuuri et al., 1996). In our study, TPA and PGE2 increased FLRG mRNA levels only after long term treatment. Therefore, in short term cultures, TPA and PGE2 increase both ßA subunit and follistatin expression, without a significant effect on FLRG mRNA levels. However, after long term treatment, TPA and PGE2 significantly increase both ßA subunit and FLRG expression, but have only a weak effect on follistatin mRNA (Tuuri et al., 1996). Furthermore, FLRG protein has been previously identified as a nuclear, as well as a secretory, protein in human granulosa cells (Tortoriello et al., 2001), and it may thus have distinct intracellular actions from those of the secreted protein, follistatin. Although both FLRG protein and follistatin prevent the exogenous (endocrine or paracrine) effects of activins, follistatin is much more potent than FLRG protein in inhibiting endogenous (autocrine) function of activins (Sidis et al., 2002). TPA and PGE2 induced follistatin and FLRG expression may thus antagonize the effect of activin on steroidogenesis at different time points.
Although the gonadotrophins FSH and LH are essential in the regulation of physiological functions of granulosa-luteal cells, our data suggest that these gonadotrophins are not critical in the regulation of FLRG expression in human granulosa-luteal cells. Gonadotrophins exert their effects on granulosa-luteal cells principally via increased intracellular cAMP formation and protein kinase A activation (Richards, 2001). Therefore, it was not surprising that bypassing the gonadotrophin receptors and activation of protein kinase A directly with (Bu)2cAMP had no significant effect on FLRG expression. A previous report has demonstrated that follistatin gene expression is rapidly (8 h) upregulated by cAMP in human granulosa-luteal cells, while the effect of TPA was clearly weaker and sometimes absent (Tuuri et al., 1996). Thus, it seems that the protein kinase A pathway is much more important than the protein kinase C pathway in the regulation of follistatin gene expression. This is clearly different from the regulation of FLRG expression, which is mainly through the protein kinase C pathway. The different effects of the protein kinase A and C signal transduction pathways on follistatin and FLRG expression may contribute (by modifying activin bioactivity) to the sometimes contradictory effects of these two kinases on granulosa-luteal cell steroidogenesis. Previous reports have demonstrated that the activation of both protein kinase A and C stimulates progesterone accumulation in human granulosa-luteal cells, but the stimulatory effect of cAMP on progesterone secretion is inhibited by TPA co-treatment (McAllister et al., 1994; Hori et al., 1998). Our results confirmed these effects, indicating that there are interactions of protein kinase A and C activities in the regulation of progesterone synthesis.
One critical factor implicated in the ovulation process and luteinization of preovulatory follicles is progesterone, which is physiologically stimulated by gonadotrophins via the activation of cAMP dependent kinases (Richards et al., 1998). Although the increase of FLRG expression by TPA and PGE2 stimulation was also associated with an elevation of progesterone secretion in our study, the unresponsiveness of FLRG mRNA levels to gonadotrophin and (Bu)2cAMP treatments suggests that FLRG expression is not always associated with the induction of progesterone secretion. This is further supported by our unpublished finding that etomidate treatment (an inhibitor of steroidogenesis) (De Coster et al., 1988) has no significant effect on the FLRG expression in granulosa-luteal cell cultures.
Taken together, our results suggest that FLRG mRNA expression in human granulosa-luteal cells is upregulated via activation of protein kinase C and by PGE2. The regulation of FLRG expression by protein kinase C activity and PGE2 may modify the local biological functions of activins in human ovaries.
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Acknowledgements |
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Ms Merja Haukka and Minna Heiskanen are thanked for their technical assistance. The company Serono-Nordic (Vantaa, Finland) is thanked for generously providing the recombinant human FSH and LH for in-vitro experiments. This work was supported by the Emil Aaltonen Foundation, Jalmari & Rauha Ahokas Foundation, Paulo Foundation, Maud Kuistila Foundation (to J.L.), Academy of Finland, Sigrid Juselius Foundation, Novo Nordisk Foundation, Pediatric Research Foundation, and Kuopio University Hospital (to R.V.).
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4 To whom correspondence should be addressed. E-mail: Jiangi.Liu{at}helsinki.fi
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References |
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