Molecular Human Reproduction, Vol. 8, No. 8, 734-741,
August 2002
© 2002 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
The soluble and membrane-anchored forms of heparin-binding epidermal growth factor-like growth factor appear to play opposing roles in the survival and apoptosis of human luteinized granulosa cells
Department of Obstetrics and Gynecology, Asahikawa Medical College, Midorigaoka Higashi 2-1-1-1, Asahikawa, Japan 078-8510
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Abstract |
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This study aims to investigate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and its role in regulating apoptosis of human luteinized granulosa cells (LGC). By using RT–PCR and immunocytochemistry, the expression of HB-EGF and the EGF receptor family was demonstrated. HER4, one of the two cognate receptors for HB-EGF, was found translocated into the nucleus. HB-EGF exists in two forms, the precursor membrane-anchored form and the mature secreted form. Addition of recombinant HB-EGF, which acts as the secreted form, into the cell culture inhibited apoptosis and appeared to stimulate mitosis, indicating that the secreted form is potentially an anti-apoptotic factor and a mitogen for LGC. In contrast, CRM197, a specific inhibitor for the interaction between HB-EGF and the EGF receptor, inhibited rather than enhanced apoptosis, suggesting that the membrane-anchored form constitutively functions as a pro-apoptotic factor for LGC. Furthermore, the finding that apoptosis inhibition by CRM197 in the aggregate cells was much more pronounced than in the single cells indicates that pro-apoptotic activity was carried out in a juxtacrine fashion, as would be expected for the membrane-anchored form of HB-EGF. These data suggest that HB-EGF may be a unique regulator of LGC apoptosis, with two functionally opposing products arising from the same gene.
apoptosis/EGF receptor family/HB-EGF/luteinized granulosa cells
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The corpus luteum plays a critical role in regulation of the menstrual cycle and is necessary for maintaining pregnancy before the luteo-placental shift. Functionally and morphologically, the corpus luteum is a very dynamic endocrine organ. Timely cessation of progesterone secretion (i.e. functional luteolysis) is required to allow a new cohort of follicles to grow and ovulate, thus maintaining the cyclic pattern of reproduction. The functionally inactive corpus luteum should be removed (i.e. structural luteolysis) to avoid accumulation of non-functional luteal tissue within the ovary. Although it is known that the mid-cycle surge of LH is the primary signal for initiation of luteinization, the structural development, integrity and control of the lifespan of the corpus luteum probably rely on other factors or intrinsic mechanisms (Stouffer, 1996
). Accumulating evidence suggests that ovarian-derived local factors such as growth factors may be involved in these processes. For example, in the developing corpus luteum, ovarian-derived vascular endothelial growth factor has been proposed as a major angiogenic factor responsible for the process of neovascularization, which is essential for normal structure and function of the mature corpus luteum (Reynolds et al., 2000).
Programmed cell death, or apoptosis, is an active cellular suicide process by which multicellular organisms delete cells that are superfluous or damaged beyond repair in order to maintain the homeostasis of the whole organism. Studies have indicated that structural luteolysis in the human is mediated, in part, by apoptosis (Shikone et al., 1996; Yuan and Giudice, 1997; Morales et al., 2000). Characteristic morphological features such as apoptotic bodies have been clearly demonstrated in luteal cells of regressing corpus luteum (Morales et al., 2000). Apoptosis can be directly triggered by death signals or induced by deprivation of survival factors (Hsu and Hsuen, 2000), but the factors that initiate apoptosis in the human corpus luteum remain unidentified.
Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a relatively recently discovered member of the EGF growth factor family and has a strong affinity for immobilized heparin (Higashiyama et al., 1991). Like other members of the EGF family of ligands, HB-EGF interacts with transmembrane proteins known as the EGF receptor family. Four members of this receptor family have been identified: HER1/erbB1 (also known as the EGF receptor), HER2/erbB2, HER3/erbB3 and HER4/erbB4 (Elenius et al., 1997). In cells, HB-EGF is first synthesized as a membrane-anchored precursor form (proHB-EGF) and then cleaved to yield the soluble mature form (sHB-EGF) through a regulated proteolytic process known as ectodomain shedding (Goishi et al., 1995). The secreted form of HB-EGF is identified as a potent mitogen for many cell types including smooth muscle cells, fibroblasts and keratinocytes. On the other hand, proHB-EGF is also biologically active. Its functions vary from stimulating cell growth and suppressing cell death to inhibiting cell growth and inducing apoptosis, depending on the type of the target cells (Raab and Klagsbrun, 1997; Iwamoto and Mekada, 2000). However, knowledge about expression of HB-EGF in the ovary is very limited (Nakamura et al., 2001), and there is no study on the functions of HB-EGF in the corpus luteum.
In this study, we present the first evidence that HB-EGF is expressed in human luteinized granulosa cells (LGC). In addition, using recombinant HB-EGF that has the same action as sHB-EGF, we show that sHB-EGF is potentially a mitogen and an inhibitor of apoptosis for LGC. Finally, using the cross-reacting material 197 (CRM197), a specific inhibitor for the interaction between HB-EGF and HER1 (Higashiyama et al., 1995; Mitamura et al., 1995), we are able to demonstrate that proHB-EGF functions constitutively to stimulate apoptosis of LGC.
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Reagents
For RNA extraction and RT–PCR, Isogen reagent was purchased from Nippon Gene (Tokyo, Japan). Moloney Murine Leukemia Virus (M-MLV) reverse transcriptase and Expand High Fidelity PCR System, an enzyme mixture containing thermostable Taq DNA and a proofreading polymerase, were purchased from Gibco BRL (Rockville, MD, USA) and Boehringer Mannheim (Mannheim, Germany) respectively. For real-time PCR, QuantiTect SYBR Green PCR kit was purchased from Qiagen Inc. (Valencia, CA, USA). For the immunocytochemical study, Vector ABC-P0 kit (rabbit IgG), AEC substrate kit for peroxidase and IgG fraction of normal rabbit serum were purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). Polyclonal antibody against HER4 (C-18) and the blocking peptide were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyclonal antibody to HB-EGF (H-1) was a generous donation from Dr Shigeki Higashiyama of Osaka University Medical School. The in-situ Apoptosis Detection Kit was purchased from TaKaRa Biomedicals (Tokyo, Japan). CRM197 was a product from Sigma Chemical Co. (St Louis, MO, USA) and recombinant human HB-EGF was purchased from R&D Systems Inc. (Minneapolis, MN, USA).
Isolation and culture of human LGC
Human LGC were isolated from follicular aspirates of patients undergoing IVF–embryo transfer treatment due to male factor or tubal obstruction. Informed consent for using the cells for experiments was obtained from all the patients. The study was also approved by the local ethics committee. The cells were derived from patients who had received a follicle-stimulation regime before oocyte retrieval, including a desensitizing protocol using GnRH agonist (buserelin acetate; Suprecur, Hoechst, Tokyo, Japan) and follicular stimulation with HMG (Pergonal; Teikokuzouki, Tokyo, Japan). HCG 10 000 IU (Mochida Pharmaceutical, Tokyo, Japan) was administered when the leading follicle was >16 mm in diameter. Oocyte retrieval was performed 34–36 h after the HCG injection.
The method of isolating LGC was similar to that previously reported, with some modifications (Matsubara et al., 2000). Collected cells were first centrifuged at 133.1 g for 5 min; then the sedimentary cells were laid onto 4 ml Ficoll-Paque (Pharmacia Biotech, Wikströms, Sweden) and centrifuged at 33.3 g for 30 min at 20°C to remove the red blood cells. Cells at the interphase were collected, washed with Hank's balanced salt solution (HBSS) free of calcium and magnesium (Gibco), and filtered through a 70 µm pore nylon mesh (Becton Dickinson Labware, Becton Dickinson and Co., Franklin Lakes, NJ, USA). Cell number was counted with a haemocytometer and cell viability was assessed by the Trypan Blue exclusion test. Cells were plated at 2x105 live cells per 60 mm dish (Becton Dickinson) and cultured at 37°C under 5% CO2 and air. The cells were initially cultured for 48 h in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (Gibco), 100 units/ml of penicillin, 100 µg/ml of streptomycin and 250 ng/ml of amphotericin B (Sigma, Irvine, UK) with one interval change of medium at 24 h. The cells were then cultured in serum-free medium supplemented with recombinant HB-EGF, CRM197 or appropriate vehicles for 24 h before analysis of apoptosis. For assessing the response of HB-EGF mRNA to recombinant HB-EGF simulation, cells were first starved of serum for 12 h after the initial culture and then treated with 10 ng/ml of recombinant HB-EGF for various periods of time.
Oil red staining and criteria for defining apoptosis
Morphology of LGC was observed by staining the nuclei with haematoxylin and simultaneously staining the lipid droplets in the cytoplasm with oil red. The method was adopted from that previously reported (Disbrey and Rack, 1970) with important modifications. After 24 h treatment with different doses of CRM197, recombinant HB-EGF or the vehicles in serum-free medium, cells were fixed in 10% PBS-buffered formalin at room temperature for 5 min. After a brief wash with distilled water, cells were stained with oil red (Nacalai Tesque INC, Kyoto, Japan) for 15 min, washed in tap water for 4 min, and then stained with Gill's Hematoxylin V (Muto Pure Chemicals Ltd, Tokyo, Japan) for 4 min. After another 4 min wash, cells were mounted in an aqueous mounting medium (Immunon, Pittsburgh, USA). Finally, the wall of each culture dish was removed with a hot blade and the cells were observed under a conventional light microscope. This method allowed both identification of luteal cells and observation of the nuclei. A cell was considered apoptotic when it exhibited apoptotic bodies containing the nuclear substances or chromatin condensation in the nuclei (Kerr et al., 1994).
Immunocytochemistry
For immunocytochemical staining, cells were fixed in 4% PBS-buffered paraformaldehyde (PFA) for 15 min at room temperature followed by treatment with 0.3% H2O2 in methanol for 20 min to inactivate the intrinsic peroxidase. After being blocked with normal goat serum peroxidase (Vector) for 20 min at room temperature, cells were incubated with primary antibodies (diluted 1:100 in PBS containing 1% bovine serum albumin) against HB-EGF (H-1) or HER4 (C-18) for 3 h at room temperature. H-1 was a rabbit polyclonal antibody generated with a synthetic peptide corresponding to COOH-terminal residues 185–208 of the HB-EGF precursor. H-1 detected proHB-EGF and did not cross-react with sHB-EGF. It was specific and did not cross-react with other members of the EGF family (Miyagawa et al., 1995). C-18 was a rabbit polyclonal antibody raised against a peptide corresponding to amino acids 1291–1308 mapping at the carboxyl terminus of human HER4 (Chow et al., 1997).
Staining was performed with the ABC method (avidin:biotinylated enzyme complex) using a Vector ABC-P0 kit (rabbit IgG) (Vector) according to the manufacturer's protocol. Briefly, incubation with the primary antibody was followed by incubation with biotinylated goat anti-rabbit IgG (the secondary antibody), and then with horseradish peroxidase-conjugated avidin. Both were carried out at room temperature for 30 min. The peroxidase activity was visualized with 3-amino-9-ethylcarbazol (AEC) using the AEC substrate kit for peroxidase (Vector). Finally, cells were either directly mounted with aqueous mounting medium (for staining of HER4) or counter-stained first with haematoxylin followed by mounting (for staining of HB-EGF). For the negative control of HB-EGF, the primary antibody was replaced with IgG fraction of normal rabbit serum at the same concentration. For the negative control of HER4, besides replacement of the primary antibody with the normal serum, an overnight preabsorption with an excess amount (5x) of blocking peptide at 4°C was performed.
RT–PCR and real-time RT–PCR
Total RNA was extracted using the Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's protocol. Extracted RNA was dissolved in 10–30 µl deionized distilled water and stored at –80°C. For RT, a 10 µl volume contained 1 µg total RNA, 0.1 µmol DTT, 0.25 µg oligo(dT)12–18, 100 IU M-MLV reverse transcriptase (Gibco) and 5 nmol of each dNTP. The reaction system was incubated at 40°C in a water bath for 3 h, heated at 95°C for 5 min to inactivate the reverse transcriptase, and then quickly chilled in ice and stored at –20°C.
PCR of the cDNA was performed in a 20 µl reaction mix containing 1 µl of the RT product, 2 µl of dimethyl sulphoxide (DMSO), 4 nmol of each dNTP, 30 nmol of MgCl2, 0.3 µl of Expand High Fidelity PCR System and 6 pmol of each primer set. All amplifications were performed with 30 cycles. The amplification cycle for of HB-EGF consisted of 94°C for 30 s, 58°C for 30 s and 72°C for 1 min. Amplification cycles for HER1, HER2, HER3 and GAPDH consisted of 94°C for 30 s, 65°C for 30 s and 72°C for 1 min. The amplification cycle for HER4 consisted of 94°C for 30 s, 60°C for 75 s and 72°C for 2 min. PCR was done with the GeneAmpTM PCR System 9600 (Perkin-Elmer, Branchburg, NJ, USA). Under these conditions, amplification of HB-EGF was established to be at the exponential stage of PCR. The primers used for the experiments are listed in Table I. All the primer sets except that for HB-EGF have been published, and their specificity was verified with restriction enzyme analysis in this study. The primer set for amplification of HB-EGF was designed by ourselves. The PCR product was confirmed by direct sequencing using the SEQ4x4 Personal Sequencing System and the Thermal Sequenase Cy 5.5 Dye Terminator Cycle Sequencing kit (Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA). PCR products were subject to electrophoresis in 1% agarose gel, stained with ethidium bromide, visualized under UV light with a UV transilluminator (LPL, Japan) and photographed with FAS-II (Toyobo, Japan).
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Real-time PCR was performed to assess the relative changes in levels of HB-EGF mRNA using the instrument and software of the Smart Cycler System (Cepheid, Sunnyvale, CA, USA). Each 25 µl PCR reaction mix consisted of 1 µl RT product, 12.5 µl of 2x QuantiTect BYBR Green PCR Master Mix (that contained HotStarTaq DNA Polymerase, dNTP mix, SYBR Green I and MgCL2), and 7.5 pmol appropriate primer sets. The primer sets used were as described above. The thermal cycling conditions were also the same as above except for an initial heating at 95°C for 15 min to activate the HotStarTaq DNA Polymerase. GAPDH was chosen as the internal standard to control for the cDNA loading and to normalize HB-EGF. The expression of GAPDH mRNA itself had been found to be relatively stable during 24 h treatment with 10 ng/ml of recombinant HB-EGF or the vehicle. For each unknown sample, concentrations of HB-EGF and GAPDH were determined from the respective standard curves.
Detection of apoptosis with in-situ 3' end-labelling of DNA
Terminal deoxynucleotide transferase (TdT)-mediated dUTP-FITC nick end-labelling (TUNEL) was performed on human LGC using the in-situ Apoptosis Detection Kit according to the manufacturer's instructions. Briefly, cells were first fixed in situ with 4% PFA for 15 min at room temperature. This was followed by inactivation of endogenous peroxidase, permeabilization, labelling with FITC-dUTP and anti-FITC HRP conjugate. The labelling was visualized with AEC, counterstained with haematoxylin, and observed under a conventional light microscope. The negative control had the TdT enzyme replaced by equivalent amounts of labelling buffer.
Statistical analysis
Cells collected from patients were randomly allocated to culture dishes. The cells in each culture dish in each independent experiment were obtained from the same patient. In the quantitative study of apoptosis, aggregate cells and single cells were counted separately. The percentage of apoptosis (incidence of apoptosis) was computed after having counted 
500 single or 1000 aggregate cells at x200 magnification from at least 10 randomly selected fields for each dish. The counting was conducted in a double-blind manner in that the dishes were coded and the observer did not know what treatment the dishes had been given. In the real-time PCR experiment, dividing the concentration of HB-EGF at each time point by that of the corresponding GAPDH resulted in a normalized HB-EGF value that was designated as relative amount of HB-EGF. Data were expressed as mean ± SD, and were analysed with a paired Student's t-test using Statview J-4.5 software. P < 0.05 was considered statistically significant.
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Expression of HB-EGF
RT–PCR detection of HB-EGF clearly showed a 439 bp band of HB-EGF mRNA in LGC (Figure 1A
). Moreover, as measured with real-time PCR, addition of 10 ng/ml recombinant HB-EGF to the culture acutely up-regulated expression of the HB-EGF transcript (Figure 1B). The highest expression of HB-EGF, found after the 2 h treatment, was nearly 2.5 times that of the control (0.19 ± 0.10 versus 0.08 ± 0.05; P < 0.01). The level was still higher at 4 h, approximately twice that of the control (0.16 ± 0.09 versus 0.08 ± 0.05; P < 0.05), but was reduced to about the pretreatment level by 24 h.
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Immunochemical staining of proHB-EGF in the LGC revealed that most LGC were immunoreactive (Figure 2A
). The nature of the staining was granular and the distribution was predominantly cytoplasmic and membranous. That the molecule is membrane-anchored was suggested by the often concentrated staining at the periphery of the cell or at the junctional interphase between cells, and by direct identification of immunoactivity in cells that happened to have folded or retracted borders. Cells were not stained when the primary antibody was replaced with IgG of normal serum (Figure 2B).
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Expression of HB-EGF receptors
RT–PCR detection showed that all the four members of the EGF receptor family are expressed in human LGC (Figure 3
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Immunolocalization of HER4 protein showed that nearly all the cells were moderately or strongly stained (Figure 4
). Distribution of immunoreactivity was predominantly nuclear with only faint staining in the cytoplasm of aggregate cells, but the nucleoli were not stained. The staining was negative when the primary antibody was replaced with normal serum or when the primary antibody that had been preincubated with excess blocking peptide was used (data not shown).
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Effects of recombinant HB-EGF and CRM197 on LGC
As shown by the oil red staining method, cells were well spread and polygonal in shape with large round nuclei and abundant cytoplasm (Figure 5
). Lipid droplets in the cytoplasm were stained red. Typically, apoptotic cells showed shrinkage of the cytoplasm, blebbing of the membrane, condensed chromatin and formation of apoptotic bodies that sometimes contained fragments of nuclear components (Figure 6). To validate the morphological criteria we were using, nucleosomal cleavage of DNA, a commonly used biochemical marker of apoptosis, was investigated by performing TUNEL. Results showed that apoptotic bodies were associated with DNA cleavage (Figure 7A). The staining was negative when TdT enzyme was replaced with the labelling buffer (Figure 7B).
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To elucidate the role of sHB-EGF in apoptosis of LGC, cells were treated with recombinant HB-EGF. As a result, the incidence of apoptosis in both single and aggregate cells decreased in a dose-dependent manner (Figure 8
). Specifically, treatment with recombinant HB-EGF at 10 and 100 ng/ml reduced the incidence of apoptosis in the single cells from 38.0 ± 7.3 to 28.2 ± 1.6 and 23.0 ± 2.1% (P < 0.05) respectively. The incidence of apoptosis in the aggregate cells decreased from 39.2 ± 6.1 to 25.2 ± 4.1 and 18.2 ± 2.8% (P < 0.05) respectively. In addition, although mitosis of LGC in the serum-free culture was extremely rare, it was occasionally found in cells treated with recombinant HB-EGF, even at the lower concentration (10 ng/ml) (Figure 9).
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To analyse the endogenous activity of HB-EGF in LGC, binding of HB-EGF with the receptor HER1 was disrupted with CRM197. This treatment markedly reduced the incidence of apoptosis in both single and aggregate cells in a dose-dependent manner (Figure 10
). CRM197 at 1 and 10 µg/ml reduced the incidence of apoptosis in the single cells from 40.1 ± 8.6 to 36.2 ± 9.7 and 28.7 ± 7.0% (P < 0.05) respectively. The effect was even more evident in the aggregate cells with the incidence of apoptosis being decreased from 37.8 ± 8.7 to 29.4 ± 11.2 and 17.6 ± 7.2% (P < 0.05) respectively. The difference between the two treatment groups in aggregate cells was also statistically significant (P < 0.05).
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To study the juxtacrine activity of proHB-EGF, the difference in the extent of apoptosis inhibition between single and aggregate cells in response to the above two treatments was analysed. The extent of apoptosis inhibition was expressed as the percentage decrease in the incidence of apoptosis. The latter was derived by first subtracting the apoptosis incidence of post-treatment from that of control and then dividing by the apoptosis incidence of the control. As a result, it was found that the aggregate cells showed more pronounced inhibition of apoptosis than the single cells in response to treatment with CRM197 at 10 µg/ml, with the percentage decrease in the incidence of apoptosis being 52.1 ± 18.3 versus 20.4 ± 15.3% (P < 0.05; Figure 11
). No similar result was found in the experiment with recombinant HB-EGF.
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Discussion |
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To our knowledge, this is the first study addressing the role of HB-EGF in the survival and apoptosis of luteal cells. HB-EGF was found to be expressed in human LGC. In addition, the HB-EGF transcript increased rapidly and transiently in response to sHB-EGF stimulation, which is consistent with findings in other cells, indicating that HB-EGF is an immediate–early gene (Dluz et al., 1993
; Polihronis et al., 1996). The finding that sHB-EGF suppressed apoptosis and appeared to stimulate mitosis of human LGC suggests that sHB-EGF is a potential anti-apoptotic factor and mitogen for these cells. Surprisingly, however, when the endogenous activity of HB-EGF was blocked with CRM197, apoptosis of human LGC, instead of being enhanced as expected, was inhibited, indicating that HB-EGF also constitutively functions as a pro-apoptotic factor. Since the two forms of HB-EGF may have different or even opposite functions depending on the cell types, we suspected that the pro-apoptotic activity may be attributable to the juxtacrine activity of proHB-EGF. To elucidate this issue, the incidence of apoptosis of LGC in single cells and aggregate cells was counted separately. The rationale was that in the context of the mechanism of proHB-EGF activities, both single and aggregate LGC in culture are subject to autocrine regulation by proHB-EGF. This appears to be true because apoptosis in single cells was inhibited by the CRM197 treatment. However, aggregate cells differ from single cells in that they are also subject to juxtacrine activity from adjacent cells. The differential response of single and aggregate cells to the CRM197 treatment reflects the juxtacrine activity of proHB-EGF. Accordingly, the fact that aggregate cells showed a much more prominent reduction in apoptosis than single cells in response to the CRM197 treatment indicates that pro-apoptotic activity of proHB-EGF is carried out in a juxtacrine fashion. Thus, the constitutive juxtacrine/autocrine action of proHB-EGF is pro apoptotic whereas the potential paracrine/autocrine action of sHB-EGF is anti-apoptotic for LGC.
However, one seeming paradox has to be explained. Since CRM197 added to the culture medium should have been able to interrupt not only pro-apoptotic activity of proHB-EGF but also anti-apoptotic activity of sHB-EGF, why was only the result of proHB-EGF interruption represented in the final outcome? We suspect that both activities of sHB-EGF and proHB-EGF are present in the cell culture, but the ultimate outcome of the CRM197 treatment or the fate of cells will depend on which activity is the most prominent or dominant. In our study, data showing that exogenous sHB-EGF effectively inhibits apoptosis suggest that in this culture condition (low cell density and serum-free medium), endogenous secretion of HB-EGF may be quite limited, and not enough to bind the EGF receptors significantly. Consequently, the juxtacrine/autocrine pro-apoptotic activity of proHB-EGF may be more prominent than the paracrine/autocrine anti-apoptotic activity of sHB-EGF, so that the net result of interruption of both activities will be the protection of cells from death. In this regard, a recent report has shown that although cells undergo constitutive ectodomain shedding of proHB-EGF in culture medium supplemented with serum, the shedding is minimized in serum-free conditions, thereby giving further credence to this hypothesis (Hirata et al., 2001).
Among the four members of the EGF receptor family, HB-EGF directly binds to two, HER1 and HER4 (Elenius et al., 1997). Although HER1 transcripts were observed, protein expression of HER1 was not investigated in this study because there have been previous reports that it is expressed in human LGC (Tekpetey et al., 1995; Almahbobi et al., 1998). However, since CRM197 is a specific inhibitor for the interaction between HB-EGF and HER1, the finding in the present study showing that CRM197 affects apoptosis of human LGC confirms the presence of HER1 in these cells. The finding also suggests that the pro-apoptotic activity of proHB-EGF is at least in part mediated through HER1.
Until now, there has been no information about the expression of HER4 in human lutein cells. We demonstrated the expression of HER4 mRNA and protein in human LGC. However, the finding that HER4 protein in these cells was localized to the nucleus is unexpected, because polypeptide growth factor receptors are generally known to be membrane proteins. This pattern of HER4 staining is unlikely to be an artefact of the method because the same polyclonal antibody, C-18, was able to stain HER4 both in the cytoplasm (Furger et al., 1998) and cell membrane (Chow et al., 1997) in other cells. Furthermore, nuclear expression of HER4 protein shown by C-18 was confirmed by a monoclonal antibody (HFR-1) that had been raised against a different epitope in the intracytoplasmic domain (Srinivasan et al., 2000). The phenomenon of nuclear targeting of polypeptide growth factors and their receptors has recently attracted attention, and has been proposed as an alternative or complimentary signalling pathway to the classic receptor-mediated signal transduction pathways (Jans, 1994; Moroianu and Riordan, 1994). The mechanism is still not clear but the nuclear localization signal (NLS) within these polypeptides is believed to be critical for the translocation (Jans and Hassan, 1998; Keresztes and Boonstra, 1999). In this respect, it is worth noting that putative NLSs exist in the HER4 molecule (Srinivasan et al., 2000). Nevertheless, no nuclear actions of HER4 in LGC are known at this time.
Transcripts for HER2 and HER3 were also investigated in this study because it is known that heterodimerization and molecular modulation between the four members of the EGF receptor family occurs (Carraway and Cantley, 1994; Carraway et al., 1997). Our RT–PCR results indicate that this possibility exists as mRNA of both HER2 and HER3 were expressed.
It is not clear what molecular differences in sHB-EGF and proHB-EGF are responsible for the opposite biological responses. Neither is it clear how HER1, a receptor known for its mitogenic and anti-apoptotic potential, can be utilized to transfer death signals. Indeed, disrupting HER1 autophosphorylation or inhibiting the MAP kinase pathway components such as MEK/MAPKK and Raf in human LGC all result in apoptosis, indicating that this pathway normally functions to support the survival of human LGC (Oliver et al., 1999; Khan et al., 2000). It is hypothesized that homodimerization/oligomerization of proHB-EGF or complex formation with other membrane proteins may be responsible for the differences. Formation of the complex would be able to induce oligomerization of HER1 that is not achieved by sHB-EGF, thereby generating a downstream signal linked to growth inhibition and apoptosis, which is qualitatively different from that generated by sHB-EGF (Iwamoto et al., 1999; Iwamoto and Mekada, 2000).
The concept that the secreted and membrane-anchored forms of HB-EGF play opposing biological roles is only recently emerging (Iwamoto et al., 1999). It has been used to explain the role of HB-EGF in the wound healing process. It is proposed that sHB-EGF stimulates the migration and growth of cells at the early stage of healing, whereas proHB-EGF causes cell growth arrest when the healing process is in completion (Iwamoto and Mekada, 2000). Whether a similar mechanism for HB-EGF works in the corpus luteum, namely that the soluble form stimulates luteal cell growth and survival during luteal development whereas the precursor form initiates apoptosis of luteal cells during regression of the corpus luteum, deserves consideration.
In conclusion, HB-EGF and its receptors were demonstrated in human LGC, and the two forms of HB-EGF were found to play opposing roles in cell apoptosis and survival. These data suggest that HB-EGF may be involved in the development and regression of corpus luteum by regulating growth, survival and apoptosis of luteal cells. Further study on HB-EGF in the corpus luteum may shed new light on the regulatory mechanism of this important organ.
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We thank Ms Kirio at our department for her efficient secretarial support. We also would like to thank Mr Shizuo Kato at the Department of Pathology of the Asahikawa Medical College hospital for his kind technical assistance.
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1 To whom correspondence should be addressed. E-mail: pan{at}mail.asahikawa-med.ac.jp
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References |
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Submitted on August 16, 2001; resubmitted on December 19, 2001; accepted on May 7, 2002.
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