Molecular Human Reproduction, Vol. 8, No. 1, 48-57,
January 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes
1 Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel and 2 Department of Obstetrics and Gynecology, Fukui Medical University, Fukui 910-1193, Japan
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Abstract |
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Human granulosa cells were immortalized by transfection of the primary cells with a mutated p53 gene in combination with the Harvey-ras oncogene, yielding established cell lines designated HGP53. Here we report that forskolin, 8-Br-cAMP and FSH modulate cell growth and steroidogenesis in HGP53 cells. Low concentrations of 8-Br-cAMP or FSH stimulated cell proliferation, while higher doses attenuated cell proliferation. Progesterone production was already evident at an FSH concentration of 0.3 mIU/ml and was maximally stimulated (50–135-fold) at 50 mIU/ml of FSH. Expression levels of steroidogenic acute regulatory protein (StAR), adrenodoxin and cytochrome P450scc were enhanced 64-, 48- and 3.1-fold respectively by FSH stimulation. Dexamethasone enhanced FSH/cAMP-induced steroidogenesis and this effect involved a marked elevation in the intracellular level of adrenodoxin and P450scc, concomitantly with a marked decrease in StAR. Conversely, basic fibroblast growth factor attenuated FSH-stimulated progesterone production, and this effect involved reductions in adrenodoxin, P450scc and StAR levels. These data suggest that the rate of steroidogenesis may be determined by the ratio of StAR and P450scc, rather than by the level of each protein alone. Whereas FSH at a low dose slightly reduced apoptosis induced by serum withdrawal from HGP53 cells, higher doses enhanced it. Dexamethasone dramatically attenuated FSH- or forskolin-enhanced apoptosis. In conclusion, FSH-dependent mechanisms of differentiation, luteinization and apoptosis can be preserved in human granulosa cells immortalized by mutated p53. Moreover, this system lends itself to studies on cross-talk between the endocrine and paracrine factors that control these processes.
FSH/gonadotrophin bioassay/granulosa cells/StAR/steroidogenesis
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Introduction |
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Human granulosa cells obtained from IVF have been extensively and intensively used for the study of gonadotrophin/cAMP-induced steroidogenesis (Veldhuis et al., 1983
; Dlugi et al., 1984; Polan et al., 1984). However, when these cells were heavily stimulated with both FSH and HCG in vivo, they ceased to divide and the life span of such cultures is limited (Breckwoldt et al., 1996). In early attempts, long-term growth and steroidogenic potential of human granulosa–lutein cells were conferred by immortalization with SV40 large T antigen (Lie et al., 1996). These cell lines showed responsiveness to cAMP, but no consistent response to HCG or FSH could be demonstrated. In another attempt, human granulosa cells were transformed with the E6 and E7 regions of human papillomavirus, but no response to FSH or LH treatment could be demonstrated (Rainey et al., 1994). Another line established from a metastatic granulosa cell carcinoma was also characterized recently, and displayed synthesis of 17ß-estradiol, thus indicating expression of the cytochrome P450 aromatase in these cells (Zhang et al., 2000). However, the steroidogenic response to either LH or FSH was modest, and did not exceed a two-fold increase over basal levels, even at a high concentration of these hormones. Immortalization of primary human granulosa cells has also been successfully achieved using triple transfection with SV40 DNA, the Harvey-ras (Ha-ras) oncogene, and a temperature-sensitive mutant of p53 (temperature-sensitive p53val135) (Hosokawa et al., 1998a). The established cell lines became highly steroidogenic when stimulated with forskolin or 8-Br-cAMP due to de-novo synthesis of the steroidogenic acute regulatory protein (StAR) and the cytochrome P450scc enzyme system, but unfortunately these cells lost responsiveness to both LH/HCG and FSH.
The tumour suppressor gene p53 plays a key role in controlling the cell cycle (Oren, 1999), by binding to DNA and transactivating the cyclin-dependent kinase inhibitor, p21(WAF1/CIP1). Another transactivation target of p53 is Mdm2, encoding a 92kDa protein which is a major negative regulator of p53 itself (Oren, 1999). Mutations in the p53 gene, which often lead to an inability to bind to DNA and to activate p27(WAF1/CIP1) and Mdm2, are frequently involved with the development of human cancers (Levine, 1997; Agarwal et al., 1998; Prives and Hall, 1999). Since some mutated forms of p53 seem to play a dominant negative role when transfected into normal cells (Michalovitz et al., 1991), we decided to examine whether co-transfection of pre-ovulatory human granulosa cells with a dominant negative mutant of p53 and with the Ha-ras oncogene, in the absence of SV40 DNA, would be sufficient to immortalize primary human granulosa cells while maintaining gonadotrophin receptivity and preserving their steroidogenic activity induced by gonadotrophin/cAMP stimulation. In the present work, we report on the establishment of human granulosa cell lines by co-transfection of pre-ovulatory cells with a temperature-sensitive mutant of p53, p53val135 (Michalovitz et al., 1990), and with the Ha-ras gene. The Ha-ras gene has been previously shown to preserve the cells steroidogenic response (Amsterdam et al., 1988; Amsterdam and Selvaraj, 1997; Keren-Tal et al., 1997), not only to forskolin and 8-Br-cAMP, but also to FSH. Moreover, these cell lines respond to other endocrine and paracrine factors, thus providing a useful model for studying the cross-talk among different signalling pathways which control the development, differentiation and death of human granulosa cells. Moreover, since these cells are very sensitive to FSH, they may provide a useful tool for bioassays of FSH in human biological fluids.
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Materials and methods |
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Plasmids
pEJ6.6 encodes activated human Ha-ras oncogene (Shih and Weinberg, 1982
). pLTRp53cGval135 contains a chimera of mouse p53 complementary DNA and genomic DNA, including introns 2–9, under the transcriptional control of a Harvey sarcoma virus long terminal repeat. It encodes a mutant protein with an alanine to valine substitution at position 135, which is temperature-sensitive, so that it possesses wild-type activity at 32°C, but not at 37°C (Michalovitz et al., 1990).
Antibodies
Antibodies against progesterone were the generous gift of Dr F.Kohen (Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel). Monoclonal mouse anti-human Mdm2 and polyclonal rabbit anti-human p21 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Anti-rabbit immunoglobulin G (IgG) labelled with fluorescein isothiocyanate (FITC) was purchased from Sigma Israel Chemical Ltd (Rehovot, Israel). P53-specific monoclonal antibodies, PAb421 (directed against human and mouse p53 antigen) and PAb248 (directed against mouse p53 antigen) were kindly provided by Dr M.Oren (Department of Molecular Cell Biology, Weizmann Institute of Science). Anti-human adrenodoxin (ADX) antibodies were generously provided by Dr W.L.Miller (University of California, San Francisco, CA, USA) and anti-human StAR antibodies, by Dr J.F.Strauss III (University of Pennsylvania Medical Center, Philadelphia, PA, USA). Both goat anti-mouse and anti-rabbit IgG coupled to horseradish peroxidase (HRP) were obtained from Biomakor (Rehovot, Israel).
Reagents
Forskolin (a potent activator of adenylate cyclase), 8-Br-cAMP and dexamethasone (DEX) were purchased from Sigma Israel Chemical Ltd. Highly purified basic fibroblast growth factor (bFGF) was generously provided by Dr A.Yayon (Department of Molecular Cell Biology, Weizmann Institute of Science). Recombinant human FSH (hFSH) was generously provided by the National Institute of Health and Dr A.F.Parlow.
Establishment of human granulosa cell lines
Transfection of granulosa cells
Granulosa cells were obtained from women undergoing IVF at Kaplan Hospital, Rehovot, Israel. Patients received a GnRH analogue in combination with FSH or HMG, followed by administration of HCG. Granulosa cells were isolated from aspirated follicular fluid after ovum retrieval. The follicular fluid was centrifuged at 300 g for 5 min to separate granulosa cells from red blood cells. The resulting pellet was resuspended and cultured in Nunc tissue culture dishes (100 mm) with Dulbecco's modified Eagle's medium/Ham's F12 (DMEM/F12) (1:1) containing 5% fetal calf serum (FCS), penicillin (100 IU/ml) and streptomycin (100 µg/ml), for 48 h (Breckwoldt et al., 1996). Primary cultures were washed three times in phosphate-buffered saline (PBS) to remove the remaining red blood cells and transfected simultaneously with 5 µg of pEJ6.6 and 5 µg of p53val135, by the calcium phosphate precipitation procedure (Keren-Tal et al., 1995).
Isolation of colonies
Densely growing foci of transformed cells were visualized and selected after 2 weeks and transferred to 24-well plastic culture dishes. After 4 days, stably growing cells were transferred to 60 mm plastic tissue culture dishes and finally cultured in 100 mm dishes. The cells were then collected to freezing vials and kept in liquid nitrogen (Keren-Tal et al., 1995; Hosokawa et al., 1998a). All experiments with the cells were performed at between 10–20 passages.
Western blot analysis
Western blots of the cell lysates were prepared as previously described (Hosokawa et al., 1998a). Blots were incubated overnight at 4°C with the appropriate first antibody [to detect p53, Mdm2, p21(WAF1/CIP1), StAR, ADX, cytochrome P450scc or actin] followed by a 1 h incubation at room temperature with goat anti-rabbit or goat anti-mouse IgG conjugated to HRP. Immuno detection was carried out by the enhanced chemiluminescence (ECL) kit (Amersham Co., Buckinghamshire, UK).
Fluorescence-activated cell sorting (FACS) analysis
Floating cells and trypsinized-attached cells of each treatment were collected and combined in order to ensure a complete representation of the cell population (Aharoni et al., 1997; Hosokawa et al., 1998b; Sasson et al., 2001). Cells were washed with cold PBS and fixed in cold methanol (–20°C) for 1 h. Subsequently cells were centrifuged, resuspended in 0.5 ml cold PBS and stained for at least 15 min with 50 µg/ml propidium iodide in presence of RNase A (100 µg/ml). Cells were then analysed in a fluorescence-activated cell sorter (FACSort; Becton Dickinson, NJ, USA). A total of 5000 events from the gated subpopulation were recorded separately. The incidence of apoptosis in the various experiments was calculated from the sub G1 fraction (peak I) revealed by the FACS analysis of the propidium iodide-labelled cells (Hosokawa et al., 1998b; Sasson et al., 2001).
Biochemical assays
Progesterone measurement
Progesterone accumulated in the culture medium was determined by radioimmunoassay, as previously described (Kohen et al., 1975; Keren-Tal et al., 1995), at the end of cell stimulation.
Protein assay
Protein was quantified by the Bradford method (Bradford, 1976).
Statistical analysis
All experiments were repeated at least three times with essentially similar results. All values were expressed as mean ± SD (n = 3). Two means were compared using Student's t-test. Where there were more than two means, significant differences between means were determined by ANOVA. The means were then analysed by Fisher's probable least-squares differences multiple comparison.
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Results |
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Screening of FSH-responsive cell lines
In order to obtain functional immortalized human granulosa cells, a primary culture of highly luteinized granulosa cells obtained from IVF patients were co-transfected with a mutated p53 (p53val135) gene and the Ha-ras gene. Colonies were isolated 2 weeks after transfection and expanded and subcloned into stable lines. The cells were elongated in shape and were often in contact through cytoplasmic extensions (Figure 1a
). Out of 17 cell lines isolated, six clones were highly responsive to FSH stimulation and released 3–10 ng of progesterone per 106 cells per 24 h in the presence of 5% FCS (Figure 2), comparable with the amount of progesterone secreted by a previously established rat granulosa cell line, rFSHR-17 (Keren-Tal et al., 1993). The non-stimulated cells produced only ~70 pg of progesterone per 106 cells per 24 h at 37°C. Cells stimulated either with hFSH (0.1–1.0 IU/ml), forskolin (5–50 µmol/l) or 8-Br-cAMP (0.1–1.0 mmol/l) became completely rounded up (Figure 1b). This morphological change in response to both FSH and cAMP is also characteristic of normal granulosa cells (Amsterdam and Rotmensch, 1987).
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Oncogenes, tumour suppressor genes and apoptosis in HGP53 cells
We have shown previously that primary human granulosa cells triply transfected with SV40 DNA, Ha-ras gene and a temperature-sensitive mutant of p53 (p53val135)(HO-23 cells) express high amounts of the mutant p53 and are highly sensitive to changes in temperature from 37–32°C with regard to the induction of the tumour suppressive effect of p53, the induction of p21(WAF1/CIP1), mdm2 and in the induction of apoptosis. Moreover, incubation of HO-23 cell lines at 32°C in the presence of forskolin leads to a further enhancement in the extent of apoptosis. In the HGP53-110 cell line, selected as a representative of cells transfected with Ha-ras and p53val135, we found much lower expression of the mutant p53 than in the HO-23 cells. Upon shifting the growth temperature of the cells from 37 to 32°C, there was no change in HGP53-110 cells in the expression of p53 and Mdm2. However, there was a significant increase in p21(WAF1/CIP1) (Figure 3A,B
). When cells were examined for induction of apoptosis by FACS analysis after labelling of the cells with propidium iodide, it was found that 25 µmol/l forskolin for 8 h at 37 or 32°C enhanced apoptosis by 2–3 fold (Figure 3C). However, no induction of apoptosis was observed following changing the growth temperature from 37 to 32°C for 8 h, in contrast to HO-23 cells where the temperature shift induced apoptosis (Hosokawa et al., 1998b). These data suggest that the intracellular concentration of the temperature-sensitive mutant of p53 is critical for the induction of apoptosis, since the lower concentrations of the p53 mutant in HGP53-110 cells were not sufficient to induce apoptosis when activated by the shift of the growth temperature from 37 to 32°C. All other FSH-responsive cell lines, HGP53-101, 102, 104 and 111, demonstrated essentially the same pattern of p53, p21(WAF1/CIP1) and mdm2 expression and the same apoptotic characteristics as those observed for HGP53-110 cells (data not shown).
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Steroidogenesis in HGP53-101 and HGP53-110 cell lines
Basal production of progesterone per 106 HGP53-110 cells per 24 h at 37°C was 66 ± 6 pg. When cells were stimulated with increasing doses of hFSH at 37°C for 24 h, formation of progesterone increased in a dose-dependent manner with an ED50 of 15 mIU/ml and reached a plateau at 50 mIU/ml, yielding ~3–4 ng progesterone per 106 cells per 24 h at 37°C. This amount of progesterone was 60-fold higher than under basal conditions (Figure 4
). When cells were stimulated with forskolin or 8-Br-cAMP, progesterone formation was also elevated in a dose-dependent manner with an ED50 of 7.0 µmol/l for forskolin and an ED50 of 0.05 mmol/l for 8-Br-cAMP. Progesterone production reached a plateau of ~5–6 ng per 106 cells per 24 h at a concentration of 15 µmol/l of forskolin or 0.25 mmol/l of 8-Br-cAMP (Figure 4).
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When cells were incubated with saturating amounts of either 8-Br-cAMP (1 mmol/l), forskolin (50 µmol/l) or FSH (3 IU/ml), a dramatic increase in the levels of ADX in mitochondria compared with that in non-stimulated cells could be demonstrated by immunofluorescence using ADX-specific antibodies (data shown only for FSH in Figure 1c,d
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To study in detail the sensitivity of one of the most steroidogenic cell lines, HGP53-101 cells were incubated with 0.3–3000 mIU/ml of recombinant hFSH for 24 h with 5% FCS (Figure 5). Just 0.3 mIU/ml of hFSH gave a significant rise (P < 0.05) in progesterone production, and this production reached a plateau at 30 mIU/ml of FSH, making this line very suitable for the bioassay of hFSH, even at low physiological concentrations.
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Cross-talk with growth factors and steroid hormones
In order to examine whether steroids and growth factors can modulate the FSH/cAMP-mediated steroidogenic response, HGP53-110 cells were stimulated for 24 h with FSH or forskolin in the presence of DEX, bFGF, insulin or estradiol (Figure 6A
). DEX enhanced FSH-stimulated progesterone production by 2 to 3-fold. In contrast, bFGF dramatically decreased FSH-stimulated steroidogenesis (to 11%), while insulin and estradiol did not show any significant effects. Augmentation of progesterone production by DEX in forskolin-stimulated cells was very modest and was evident only at 10 nmol/l DEX, while the decrease in progesterone production by bFGF was much more mild (only up to 50%) compared with its effect on FSH-stimulated cells (Figure 6B). Insulin, even at 1 µg/ml, did not significantly reduce forskolin-stimulated progesterone production and estradiol was also without effect. DEX, bFGF, insulin and estradiol by themselves did not stimulate progesterone production (data not shown). Essentially similar results were obtained with HGP53-101 cells. It should be noted that the steroidogenic response decreased after >20 passages by 2–4 fold, but the relative responses to forskolin, 8-Br-cAMP, hFSH, DEX and bFGF remained essentially the same. However, the steroidogenic response of all HGP53 cell lines was stable, even after several cycles of freezing in liquid nitrogen and thawing, and thus they could be stored for prolonged periods without losing their sensitivity to FSH.
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Intracellular levels of StAR, ADX and cytochrome P450scc
We next examined whether enhancement of steroidogenesis is controlled by de-novo synthesis of StAR and the cytochrome P450scc enzyme system. Western blot analysis was performed on cell lysates of HGP53-110 cells, 24 h after stimulation with FSH alone, or in combination with DEX, bFGF, insulin or estradiol, and specific antibodies to human StAR, ADX and P450scc were used (Figure 7
). A concentration of 1 IU/ml of FSH increased StAR, ADX and P450scc levels by 64-, 48- and 3.1-fold respectively. Interestingly, while DEX augmented ADX levels by 2.2- and 2.5- fold at 10 and 100 nmol/l respectively (compared with FSH alone), DEX at a similar concentration reduced the intracellular levels of StAR to 0.21- and 0.23-fold respectively, suggesting that enhancement of progesterone production by DEX is mainly due to up-regulation of the cytochrome P450scc enzyme system, and occurs despite the down-regulation of StAR. The parallel 1.4- and 1.6-fold increase in the cytochrome P450scc levels by 10 and 100 nmol/l DEX respectively in the presence of FSH supports this notion. Compared with FSH stimulation alone, bFGF reduced StAR, cytochrome P450scc and ADX levels to 0.35-, 0.53- or 0.72-fold respectively. Insulin and estradiol did not show a marked effect on StAR, ADX or cytochrome P450scc (data not shown). Essentially similar results were obtained with HGP53-101 cells. It should be noted that the absolute levels of ADX and cytochrome P450scc could not be measured by the densitometer tracing due to the different affinities of the two antisera. However, the fluctuation of the intensities in the level of both ADX and cytochrome P450scc in response to the different treatments show the same tendency. The sharper increase in ADX compared with that for cytochrome P450scc is probably due to more rapid induction in ADX compared with the cytochrome P450scc in the immortalized granulosa cell lines (Hanukoglu et al., 1990). A dramatic increase in StAR, ADX and cytochrome P450scc was also detected in HGP53 cell lines following 24 h of incubation with 50 µmol/l forskolin at 37°C.
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Cell growth and apoptosis
When HGP53-110 cells were grown in 5% FCS, there was an almost linear rate of growth up to 48 h, with a 6-fold increase in cell number followed by a sharp decrease during 48–72 h of growth (Figure 8A
). When cells were incubated with increasing doses of hFSH for 24 h, an increase in cell number occurred at 3–10 mIU/ml of FSH followed by a decrease in cell number at a concentration range of 0.03–3 IU/ml. When cells were incubated with increasing concentrations of 8-Br-cAMP, an increase in cell number occurred at concentrations of 10–30 µmol/l, followed by decrease in cell number at 0.1–1mmol/l of 8-Br-cAMP.
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Since low doses of FSH increased HGP53-110 cell number, and high doses decreased the cell population, we examined whether this phenomenon involves modulation in programmed cell death caused by serum withdrawal. While 0.01 IU/ml of hFSH modestly decreased cell death following 12 h incubation, 0.1 and 1 IU/ml of the hormone gave a clear rise in programmed cell death (sub-G1 fraction of cells by FACS analysis) (Figure 9
). A much more pronounced degree of apoptosis could be induced by 5 and 50 µmol/l of forskolin. Both FSH- and forskolin- induced apoptosis could be effectively blocked by 100 nmol/l of DEX. These observations are in line with our previous observation that DEX attenuates forskolin-induced apoptosis in HO-23 immortalized human granulosa cells in a dose-dependent manner (Sasson et al., 2001).
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Discussion |
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In the present study, we demonstrate that transfection of human granulosa cells from IVF patients with a temperature-sensitive mutant of p53 (p53val135) and Ha-ras generates immortalized cells which maintain their steroidogenic response to FSH/cAMP. It was previously demonstrated that immortalization of granulosa cells without Ha-ras leads to complete de-differentiation of the cells (Amsterdam et al., 1988
), but earlier attempts also showed that transfection of the primary granulosa cells with Ha-ras alone is not sufficient to immortalize the granulosa cells (Amsterdam and Keren-Tal, unpublished data). The effect of mutated p53 (p53val135) on immortalization is probably exerted by its dominant negative effect on the endogenous wild-type p53, which in a mixed tetramer loses the ability to bind to DNA (Michalovitz et al., 1991). In addition, the expression of the mutated p53 may attenuate the endogenous expression of the wild-type p53 (Keren-Tal et al., 1995). Furthermore, a genetic instability resulting from the inactivation of p53 may facilitate events that lead to telomerase expression (Vaziri and Benchimol, 1999). Thus, it is most likely that the immortalized human granulosa cells may show high activity of telomerase, generally evident in human immortalized cells (Colgin and Reddel, 1999).
Interestingly, shifting the temperature of growth in HGP53-110 cells did not induce Mdm2 expression and apoptosis, but in contrast, induced p21 expression. Since the amount of the temperature-sensitive mutant of p53 expressed in HGP53 cell lines was much lower than in HO-23 cells, where the temperature shift was very effective in induction of both Mdm2 and apoptosis, we can suggest that: (i) induction of Mdm2 depends at least in part on the amount of the activated p53 and (ii) Mdm2 activation, rather than p21, is mainly involved in transducing the apoptotic signals exerted by p53. Indeed, it has been previously demonstrated that bFGF negates the p53-induced signal for apoptosis by up-regulation of Mdm2 (Shaulian et al., 1997; Hosokawa et al., 1998b; Amsterdam et al., 1999). On the other hand, cAMP enhanced p53-induced apoptosis involves down-regulation of Mdm2 (Hosokawa et al., 1998b).
The effect of FSH/cAMP on granulosa cell growth is not completely understood. On one hand they are considered as survival factors (Chun et al., 1994), but on the other hand an excess of cAMP can induce apoptosis in both primary and immortalized granulosa cells (Aharoni et al., 1995; Keren-Tal et al., 1995; Hosokawa et al., 1998b). The experiments performed on the effect of increasing doses of 8-Br-cAMP or FSH in the present study suggested that at a low concentration of FSH/cAMP the rate of cell growth is enhanced, while at a higher concentration of FSH/cAMP the rate of cell growth is reduced, suggesting that the effect of FSH is pronounced more on cell differentiation and luteinization. In suboptimal conditions, high levels of FSH can even lead to apoptosis.
Our data on the opposite effect of low and high doses of cAMP on granulosa cell growth are consistent with earlier observations that low doses of cAMP cause an increase in the incorporation of [3H] thymidine into DNA in isolated granulosa cells (Yong et al., 1992), while higher doses are more effective in induction of granulosa cell differentiation, luteinization and apoptosis (Aharoni et al., 1995; Keren-Tal et al., 1995).
HGP53 cells responded to FSH, forskolin or 8-Br-cAMP in the production of progesterone in a dose-dependent manner. An ~100-fold increase in progesterone production following forskolin or 8-Br cAMP appeared to be due to de-novo synthesis of StAR and the P450scc enzyme system. The somewhat lower response to FSH (~55-fold increase) is probably due to a desensitization phenomenon, which has been clearly demonstrated in both primary and FSH-responsive immortalized granulosa cells (Zor et al., 1976; Amsterdam et al., 1979; Hunzicker-Dunn and Birnbaumer, 1985; Aharoni et al., 1995; Keren-Tal et al., 1996). However, the ED50 of the response to FSH at 15 mIU/ml and the magnitude of response in progesterone production make these cells much more responsive to FSH compared with any immortalized human or rat granulosa cells, apart from those transfected with a recombinant FSH receptor (Keren-Tal et al., 1993; Rainey et al., 1994; Lie et al., 1996; Zhang et al., 2000; Nishi et al., 2001). This may be due to the fact that these cells were not transfected with SV40 DNA, known to express the T antigen, which most probably leads to de-differentiation (Amsterdam et al., 1988). The use of a temperature-sensitive mutant p53 (Michalovitz et al., 1990) plus Ha-ras, for the first time in establishing human granulosa cell lines may also prove to be useful for the immortalization of other types of endocrine cells with minimal loss of their differentiation potential. The rounding of the cells subsequent to FSH/cAMP stimulation is probably due to the increase of intracellular levels of cAMP, characteristic of both primary and immortalized FSH-responsive granulosa cells (Amsterdam et al., 1989; Keren-Tal et al., 1993). The production of progesterone in this cell line suggests that they are able to luteinize in vitro, in contrast with COV434 immortalized human granulosa cells that were established from a primary human granulosa cell tumour (Zhang et al., 2000).
The pronounced increase in StAR (Stocco and Clark, 1996; Strauss et al., 1999) expression observed in HGP53 cells probably plays a central role in the dramatic up-regulation of steroidogenesis in HGP53 cell lines stimulated by FSH/cAMP. The pronounced stimulation of progesterone production by FSH allowed us to follow the modulation of expression of StAR, P450scc and ADX in HGP53 cell lines, which serve as additional markers for the cytochrome P450scc enzyme system. Moreover, the responsiveness of the cells to the combined treatment of FSH and with either DEX or bFGF permitted us to measure intracellular levels of StAR, P450scc and ADX during modulation of steroidogenesis. The increase in StAR and the P450scc enzyme system by FSH is in line with the known effect of FSH/cAMP on primary granulosa cells (Hsueh et al., 1984; Miller, 1988; Amsterdam et al., 1989; Strauss et al., 1999). However, the enhancement of progesterone production by DEX revealed an interesting and novel phenomenon since it gave rise to P450scc and ADX expression, but attenuated StAR expression. These data are in line with a recent observation that DEX decreases StAR expression in pre-ovulatory rat follicles, but in contrast to the present study, does not change P450scc which results in reduction in progesterone production (Huang and Shirley, 2001).
Taken together, this apparent discrepancy may suggest that the rate-controlling determinant of steroidogenesis is not merely the absolute amount of either StAR or P450scc system, but probably the ratio of these factors. Thus, the decrease in progesterone production in the rat pre-ovulatory follicles (Huang and Shirley, 2001) was due to a decrease in StAR with no change in P450scc, while in the immortalized granulosa cells characterized in this study, the increase in progesterone synthesis was associated with a similar decrease in StAR, but a contrary pronounced increase in the P450scc system. In a previous study, we demonstrated a synergistic effect of forskolin and DEX in enhancement of progesterone production, but no attenuation of StAR expression induced by DEX in the forskolin-stimulated cells was noticed (Sasson et al., 2001). However, it should be noted that these observations were recorded in an immortalized human granulosa cell line transfected with SV40, Ha-ras and the temperature-sensitive mutant of p53 using 50 µmol/l of forskolin. When lower concentrations of forskolin (<15 µmol/l) were used, the suppressive effect of DEX on StAR expression was noticed (Sasson and Amsterdam, unpublished data) suggesting that high intracellular levels of cAMP may abrogate the suppressive effect of glucocorticoids on StAR expression. The mechanism of glucocorticoid-induced down-regulation of StAR is not completely understood and it may affect the rate of either its synthesis or degradation. DEX on one hand may reduce the intracellular levels of cAMP in target cells (Borski, 2000) and thus may attenuate the de-novo synthesis of StAR as well as that of the p450scc enzyme system, although this was not evident in the present work. On the contrary, the P450scc enzyme system was further elevated by DEX in the presence of FSH. Therefore, a more reasonable hypothesis to be considered is that DEX may specifically modulate the half-life of StAR (Arakane et al., 1998) which was recently demonstrated to be degraded by the proteasome proteolytic system (Tajima et al., 2001).
The inhibition of progesterone production by bFGF is in line with our previous finding that in immortalized human granulosa cells, bFGF attenuates steroidogenesis (Hosokawa et al., 1998a). In this work, we clearly indicate, for the first time, that this is due to a co-ordinated attenuation of both intracellular StAR and ADX. The attenuation of StAR intracellular levels by bFGF may be due to the activation of mitogen-activated protein kinase (MAPK) by this growth factor, as it was recently demonstrated that the extracellular signal-regulated kinase (ERK) signalling cascade inhibits gonadotrophin-stimulated StAR expression (Seger et al., 2001). The decrease in the p450scc system expression in the presence of bFGF is still obscure and open for future research.
The novel immortalized human granulosa cell lines reported here can potentially serve as sensitive systems for hFSH bioassay, for the study of the regulation of growth, luteinization and programmed cell death, and for a detailed analysis of the regulation of steroidogenesis by FSH and by steroid hormones and growth factors that cross-talk with the FSH/cAMP signalling pathway.
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Acknowledgements |
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We would like to thank Dr M.Liscovitch and Dr M.Walker for helpful discussion and Drs M.Oren, W.L.Miller, J.F.Strauss III and F.Kohen for the generous supply of antibodies to p53, ADX, StAR and progesterone respectively, and Dr A.Yayon for the generous supply of bFGF. The work was supported in part by grants from the Yad Abraham Research Center for Cancer Diagnostics and Therapy and the Levin Center for Applied Research at the Weizmann Institute of Science, Rehovot, Israel. A.A. is the incumbent of the Joyce and Ben B.Eisenberg Professorial Chair of Molecular Endocrinology and Cancer Research at the Weizmann Institute of Science.
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Notes |
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3 To whom correspondence should be addressed. E-mail: abraham.amsterdam{at}weizmann.ac.il
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References |
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Agarwal, M.L., Taylor, W.R., Chernov, M.V., Chernova, O.B. and Stark, G.R. (1998) The p53 network. J. Biol. Chem., 273, 1–4.
Aharoni, D., Dantes, A., Oren, M. and Amsterdam, A. (1995) cAMP-mediated signals as determinants for apoptosis in primary granulosa cells. Exp. Cell Res., 218, 271–282.[ISI][Medline]
Aharoni, D., Meiri, I., Atzmon, R., Vlodavsky, I. and Amsterdam, A. (1997) Differential effect of components of the extracellular matrix on differentiation and apoptosis. Curr. Biol., 7, 43–51.[ISI][Medline]
Amsterdam, A. and Rotmensch, S. (1987) Structure–function relationships during granulosa cell differentiation. Endocrinol. Rev., 8, 309–337.[ISI][Medline]
Amsterdam, A. and Selvaraj, N. (1997) Control of differentiation, transformation, and apoptosis in granulosa cells by oncogenes, oncoviruses, and tumor suppressor genes. Endocrinol. Rev., 18, 435–461.
Amsterdam, A., Rotmensch, S. and Ben-Ze'ev, A. (1989) Coordinated regulation of morphological and biochemical differentiation in a steroidogenic cell: the granulosa cell model. Trends Biochem. Sci., 14, 377–382.[ISI][Medline]
Amsterdam, A., Nimrod, A., Lamprecht, S.A., Burstein, Y. and Lindner, H.R. (1979) Internalization and degradation of receptor-bound hCG in granulosa cell cultures. Am. J. Physiol., 236, E129–138.
Amsterdam, A., Zauberman, A., Meir, G., Pinhasi-Kimhi, O., Suh, B.S. and Oren, M. (1988) Cotransfection of granulosa cells with simian virus 40 and Ha-RAS oncogene generates stable lines capable of induced steroidogenesis. Proc. Natl Acad. Sci. USA, 85, 7582–7586.
Amsterdam, A., Gold, R.S., Hosokawa, K., Yoshida, Y., Sasson, R., Jung, Y. and Kotsuji, F. (1999) Crosstalk among multiple signaling pathways controlling ovarian cell death. Trends Endocrinol. Metab., 10, 255–262.[ISI][Medline]
Arakane, F., Kallen, C.B., Watari, H., Foster, J.A., Sepuri, N.B., Pain, D., Stayrook, S.E., Lewis, M., Gerton, G.L. and Strauss, J.F. (1998) The mechanism of action of steroidogenic acute regulatory protein (StAR). StAR acts on the outside of mitochondria to stimulate steroidogenesis. J. Biol. Chem., 273, 16339–16345.
Borski, R.J. (2000) Nongenomic membrane actions of glucocorticoids in vertebrates. Trends Endocrinol. Metab., 11, 427–436.[ISI][Medline]
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248–254.[ISI][Medline]
Breckwoldt, M., Selvaraj, N., Aharoni, D., Barash, A., Segal, I., Insler, V. and Amsterdam, A. (1996) Expression of Ad4-BP/cytochrome P450 side chain cleavage enzyme and induction of cell death in long-term cultures of human granulosa cells. Mol. Hum. Reprod., 2, 391–400.
Chun, S.Y., Billig, H., Tilly, J.L., Furuta, I., Tsafriri, A. and Hsueh, A.J. (1994) Gonadotropin suppression of apoptosis in cultured preovulatory follicles: mediatory role of endogenous insulin-like growth factor I. Endocrinology, 135, 1845–1853.[Abstract]
Colgin, L.M. and Reddel, R.R. (1999) Telomere maintenance mechanisms and cellular immortalization. Curr. Opin. Genet. Dev., 9, 97–103.[ISI][Medline]
Dlugi, A.M., Laufer, N., Polan, M.L., DeCherney, A.H., Tarlatzis, B.C., MacLusky, N.J. and Behrman, H.R. (1984) 17 beta-estradiol and progesterone production by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization. J. Clin. Endocrinol. Metab., 59, 986–992.[Abstract]
Hanukoglu, I., Suh, B.S., Himmelhoch, S. and Amsterdam, A. (1990) Induction and mitochondrial localization of cytochrome P450scc system enzymes in normal and transformed ovarian granulosa cells. J. Cell Biol., 111, 1373–1381.
Hosokawa, K., Dantes, A., Schere-Levy, C., Barash, A., Yoshida, Y., Kotsuji, F., Vlodavsky, I. and Amsterdam, A. (1998a) Induction of Ad4BP/SF-1, steroidogenic acute regulatory protein, and cytochrome P450scc enzyme system expression in newly established human granulosa cell lines. Endocrinology, 139, 4679–4687.
Hosokawa, K., Aharoni, D., Dantes, A., Shaulian, E., Schere-Levy, C., Atzmon, R., Kotsuji, F., Oren, M., Vlodavsky, I. and Amsterdam, A. (1998b) Modulation of Mdm2 expression and p53-induced apoptosis in immortalized human ovarian granulosa cells. Endocrinology, 139, 4688–4700.
Hsueh, A.J., Adashi, E.Y., Jones, P.B. and Welsh, T.H., Jr. (1984) Hormonal regulation of the differentiation of cultured ovarian granulosa cells. Endocrinol. Rev., 5, 76–127.[ISI][Medline]
Huang, T.J. and Shirley Li, P. (2001) Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles. Biol. Reprod., 64, 163–170.
Hunzicker-Dunn, M. and Birnbaumer, L. (1985) The involvement of adenylyl cyclase and cyclic AMP-dependent protein kinases in luteinizing hormone actions. In Ascoli, M. (ed) Luteinizing Hormone Action and Receptors, CRC Press, Boca Raton, FL, pp. 57–134.
Keren-Tal, I., Dantes, A. and Amsterdam, A. (1996) Activation of FSH-responsive adenylate cyclase by staurosporine: role for protein phosphorylation in gonadotropin receptor desensitization. Mol. Cell. Endocrinol., 116, 39–48.[ISI][Medline]
Keren-Tal, I., Dantes, A., Sprengel, R. and Amsterdam, A. (1993) Establishment of steroidogenic granulosa cell lines expressing follicle stimulating hormone receptors. Mol. Cell. Endocrinol., 95, R1–10.[ISI][Medline]
Keren-Tal, I., Suh, B.S., Dantes, A., Lindner, S., Oren, M. and Amsterdam, A. (1995) Involvement of p53 expression in cAMP-mediated apoptosis in immortalized granulosa cells. Exp. Cell Res., 218, 283–295.[ISI][Medline]
Keren-Tal, I., Dantes, A., Plehn-Dujowich, D. and Amsterdam, A. (1997) Association of Ad4BP/SF-1 transcription factor with steroidogenic activity in oncogene-transformed granulosa cells. Mol. Cell. Endocrinol., 127, 49–57.[ISI][Medline]
Kohen, F., Bauminger, S. and Lindner, H.R. (1975) Preparation of antigenic steroid-protein conjugates. In Cameron, E.H.D., Hillier, S.G. and Griffiths, K. (eds) Steroid Immunoassay, Alpha Omega Publishing Ltd, Cardiff, Wales, pp. 11–23.
Levine, A.J. (1997) p53, the cellular gatekeeper for growth and division. Cell, 88, 323–331.[ISI][Medline]
Lie, B.L., Leung, E., Leung, P.C. and Auersperg, N. (1996) Long-term growth and steroidogenic potential of human granulosa-lutein cells immortalized with SV40 large T antigen. Mol. Cell. Endocrinol., 120, 169–176.[ISI][Medline]
Michalovitz, D., Halevy, O. and Oren, M. (1990) Conditional inhibition of transformation and of cell proliferation by a temperature-sensitive mutant of p53. Cell, 62, 671–680.[ISI][Medline]
Michalovitz, D., Halevy, O. and Oren, M. (1991) p53 mutations: gains or losses? J. Cell. Biochem., 45, 22–29.[ISI][Medline]
Miller, W.L. (1988) Molecular biology of steroid hormone synthesis. Endocrinol. Rev., 9, 295–318.[ISI][Medline]
Nishi, Y., Yanase, T., Mu, Y.M., Oba, K., Ichino, I., Saito, M., Nomura, M., Mukasa, C., Okabe, T., Goto, K., Takayanagi, R., Kashimura, Y., Haji, M. and Nawata, H. (2001) Establishment and characterization of a steroidogenic human granulosa-like tumor cell line, KGN, that expresses functional follicle-stimulating hormone receptor. Endocrinology, 142, 437–445.
Oren, M. (1999) Regulation of the p53 tumor suppressor protein. J. Biol. Chem., 274, 36031–36034.
Polan, M.L., Laufer, N., Dlugi, A.M., Tarlatzis, B.C., Haseltine, F.P., DeCherney, A.H. and Behrman, H.R. (1984) Human chorionic gonadotropin and prolactin modulation of early luteal function and luteinizing hormone receptor-binding activity in cultured human granulosa-luteal cells. J. Clin. Endocrinol. Metab., 59, 773–779.[Abstract]
Prives, C. and Hall, P.A. (1999) The p53 pathway. J. Pathol., 187, 112–126.[ISI][Medline]
Rainey, W.H., Sawetawan, C., Shay, J.W., Michael, M.D., Mathis, J.M., Kutteh, W., Byrd, W. and Carr, B.R. (1994) Transformation of human granulosa cells with the E6 and E7 regions of human papillomavirus. J. Clin. Endocrinol. Metab., 78, 705–710.[Abstract]
Sasson, R., Tajima, K. and Amsterdam, A. (2001) Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2. Endocrinology, 142, 802–811.
Seger, R., Hanoch, T., Rosenberg, R., Dantes, A., Merz, W.E., Strauss, J.F. and Amsterdam, A. (2001) The ERK signaling cascade inhibits gonadotropin-stimulated steroidogenesis. J. Biol. Chem., 276, 13957–13964.
Shaulian, E., Resnitzky, D., Shifman, O., Blandino, G., Amsterdam, A., Yayon, A. and Oren, M. (1997) Induction of Mdm2 and enhancement of cell survival by bFGF. Oncogene, 15, 2717–2725.[ISI][Medline]
Shih, C. and Weinberg, R.A. (1982) Isolation of a transforming sequence from a human bladder carcinoma cell line. Cell, 29, 161–169.[ISI][Medline]
Stocco, D.M. and Clark, B.J. (1996) Regulation of the acute production of steroids in steroidogenic cells. Endocrinol. Rev., 17, 221–244.[ISI][Medline]
Strauss, J.F. III, Kallen, C.B., Christenson, L.K., Watari, H., Devoto, L., Arakane, F., Kiriakidou, M. and Sugawara, T. (1999) The steroidogenic acute regulatory protein (StAR): a window into the complexities of intracellular cholesterol trafficking. Recent Prog. Horm. Res., 54, 394–395.
Tajima, K., Babich, S., Yoshida, Y., Dantes, A., Strauss, J.F. III and Amsterdam, A. (2001) The proteasome inhibitor MG132 promotes accumulation of the steroidogenic acute regulatory protein (StAR) and steroidogenesis. FEBS Lett., 490, 59–64.[ISI][Medline]
Vaziri, H. and Benchimol, S. (1999) Alternative pathways for the extension of cellular life span: inactivation of p53/pRb and expression of telomerase. Oncogene, 18, 7676–7680.[ISI][Medline]
Veldhuis, J.D., Klase, P.A., Sandow, B.A. and Kolp, L.A. (1983) Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory Graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin. J. Clin. Endocrinol. Metab., 57, 87–93.[Abstract]
Yong, E.L., Baird, D.T. and Hillier, S.G. (1992) Mediation of gonadotrophin-stimulated growth and differentiation of human granulosa cells by adenosine-3',5'-monophosphate: one molecule, two messages. Clin. Endocrinol. (Oxf.), 37, 51–58.[Medline]
Zhang, H., Vollmer, M., De Geyter, M., Litzistorf, Y., Ladewig, A., Durrenberger, M., Guggenheim, R., Miny, P., Holzgreve, W. and De Geyter, C. (2000) Characterization of an immortalized human granulosa cell line (COV434). Mol. Hum. Reprod., 6, 146–153.
Zor, U., Lamprecht, S.A., Misulovin, Z., Koch, Y. and Lindner, H.R. (1976) Refractoriness of ovarian adenylate cyclase to continued hormonal stimulation. Biochem. Biophys. Acta, 428, 761–765.[Medline]
Submitted on June 1, 2001; accepted on October 25, 2001.
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