Comparative effects of heparin-binding epidermal growth factor-like growth factor on the growth of cultured human uterine leiomyoma cells and myometrial cells

Jiayin Wang, Noriyuki Ohara, Shigeki Takekida, Qin Xu and Takeshi Maruo1

Department of Obstetrics and Gynecology, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-Cho, Chuo-Ku, Kobe, 650-0017, Japan

1 To whom correspondence should be addressed. Email: maruo{at}kobe-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The objective of this study was to investigate the comparative effects of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on the growth of cultured human leiomyoma cells and myometrial cells. METHODS: Isolated cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml). These cells were used for immunocytochemical analysis for Ki67, western blot analysis for proliferating cell nuclear antigen (PCNA) and human EGF receptor (HER1), and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling (TUNEL) assay. RESULTS: Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate of cultured leiomyoma cells and myometrial cells. Treatment with HB-EGF also resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase in PCNA expression in cultured myometrial cells was noted following treatment with HB-EGF at concentrations >1 ng/ml, whereas an increase in PCNA expression in cultured leiomyoma cells was noted following treatment with HB-EGF at concentrations >10 ng/ml. HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells. A significant increase in HER1 expression in myometrial cells was observed when treated with HB-EGF at concentrations >10 ng/ml, whereas a significant increase in HER1 expression in leiomyoma cells was noted only by the treatment with HB-EGF at concentrations >100 ng/ml. Treatment with HB-EGF decreased the TUNEL-positive rate of those cells with no significant differences between the two cell types. CONCLUSIONS: The results obtained suggest that HB-EGF plays a role in stimulating the proliferation of leiomyoma cells and myometrial cells and in inhibiting apoptosis of those cells through augmentation of HER1 expression. Since the proliferative potential of myometrial cells responded better to HB-EGF than that of leiomyoma cells, HB-EGF may play a more vital role in myometrial growth than leiomyoma growth.

Key words: apoptosis/epidermal growth factor receptor/heparin-binding epidermal growth factor-like growth factor/leiomyoma/myometrium/proliferating cell nuclear antigen


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Uterine leiomyoma is an ovarian sex steroid hormone-dependent smooth muscle tumour. Accumulating data have demonstrated that several local growth factors are involved in leiomyoma growth, including epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), and transforming growth factor-{beta} (TGF-{beta}) (Maruo et al., 2004Go).

The EGF family includes EGF, heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor-{alpha} (TGF-{alpha}), amphiregulin, betacellulin, epiregulin, neuregulins, and neuregulin-2S (Riese and Stein, 1998Go). HB-EGF is a 22 kDa protein that was first identified in the macrophage-like cell-conditioned medium (Higashiyama et al., 1991Go). HB-EGF is synthesized as a transmembrane protein (proHB-EGF) that can be cleaved proteolytically to release mature soluble HB-EGF (sHB-EGF) (Higashiyama et al., 1992Go; Ono et al., 1994Go; Goishi et al., 1995Go). HB-EGF is a potent autocrine/paracrine mitogen for fibroblasts, smooth muscle cells, keratinocytes and endometrial stromal cells (Higashiyama et al., 1991Go; Marikovsky et al., 1993Go; Chobotova et al., 2002Go), as well as a chemoattractant for smooth muscle cells (Higashiyama et al., 1993Go). HB-EGF binds to its cognate receptors, human EGF receptor 1 (HER1) and HER4 (Elenius et al., 1997Go), as well as cell surface heparin sulphate proteoglycan via its heparin-binding domain (Higashiyama et al., 1993Go; Thompson et al., 1994Go). HB-EGF activates HER1 and HER4 (Elenius et al., 1997Go). HB-EGF stimulates the proliferation and chemotaxis in cells expressing HER1, but not proliferative response in cells expressing HER4 (Klaus et al., 1997Go).

HB-EGF mRNA has been shown to be present in myometrium and leiomyoma tissues, and to decline during the transformation of myometrial cells into leiomyoma cells (Mangrulkar et al., 1995Go). Several studies have demonstrated the presence of erbB1 mRNA (Yeh et al., 1991Go) and immunoreactive HER1 protein (Leone et al., 1991Go; Shimomura et al., 1998Go; Dixon et al., 2000Go) in leiomyoma cells and myometrial cells. These findings suggest that HB-EGF may play an important role in the growth of both myometrial cells and leiomyoma cells in an autocrine/paracrine manner. However, the physiological role of HB-EGF on these cells remains largely unknown.

In the present study, we investigated the effects of HB-EGF on the expression of Ki67, proliferating cell nuclear antigen (PCNA) and HER1, and apoptosis in cultured human leiomyoma cells and myometrial cells by immunocytochemistry, western blot analysis, and terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate (dUTP) nick-end labelling (TUNEL) assay.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Materials
Phenol Red-free Dulbecco's modified Eagle's medium (DMEM) and antibiotics (1 x 105 IU/l penicillin, 50 mg/l streptomycin) were purchased from Life Technologies (USA). Fetal bovine serum (FBS) was obtained from Sigma Chemical Co. (USA). Collagenase was obtained from Wako Pure Biochemical Industries (Osaka, Japan). Recombinant HB-EGF was obtained from R&D Systems, Inc. (USA). A mouse monoclonal antibody to PCNA (PC10, sc-56) and a rabbit polyclonal antibody to EGF receptor, HER1 (1005, sc-03), were purchased from Santa Cruz Biotechnology, Inc. (USA).

Tissue collection and cell culture
Uterine leiomyoma tissues and adjacent myometrium were obtained from pre-menopausal women with regular menstrual cycles who underwent hysterectomy for uterine leiomyomas at Kobe University Hospital. The study protocol for the collection of surgical specimens was approved by the Institutional Review Board. Informed consent was obtained from each patient before surgery for the use of uterine tissues for the present study. Patients had received no hormonal therapy for ≥6 months before surgery. The age of patients ranged from 35 to 42 years, with a mean age of 37.4 years. Each uterine specimen was examined by a pathologist for histological evaluation. Endometrial tissues were obtained from the extirpated uterus, and the day of the menstrual cycle was determined by endometrial histological dating according to the method of Noyes et al. (1950)Go. Four samples each were collected during the proliferative phase of the menstrual cycle and the secretory phase of the menstrual cycle respectively.

Leiomyoma tissues and adjacent myometrium were dissected from endometrial cell layers, washed in phosphate-buffered saline (PBS), cut into small pieces, and digested in 0.2% collagenase at 37 °C for 3–5 h. Leiomyoma cells and myometrial cells were collected by centrifugation at 460 g for 5 min and washed three times with PBS containing 1% antibiotic solution respectively. Cell viability was determined by Trypan Blue exclusion test. Isolated cells were subcultured at 37 °C for 120 h in a humidified atmosphere of 5% CO2–95% air in Phenol Red-free DMEM supplemented with 10% FBS. The monolayer cultures at ~60% confluence were stepped down to serum-free conditions for an additional 24 and 48 h in the presence or absence of graded concentrations of HB-EGF (0.1, 1, 10 and 100 ng/ml).

Immunocytochemical staining for Ki67
Cultured cells were fixed in 90% ethanol and washed with PBS three times. The fixed cells were subjected to immunostaining by the avidin/biotin immunoperoxidase method using a polyvalent immunoperoxidase kit (Omnitags, USA). A goat polyclonal antibody against HB-EGF (M-18, sc-1414; Santa Cruz Biotechnology, Inc., USA) and a mouse monoclonal antibody against Ki67 (NCL-Ki67-MM1; Novocastra Laboratory Ltd, UK) were used as the primary antibodies at the dilution of 1:100 respectively. To ensure the specificity of the immunological reaction, the cultured cells were subjected to the same immunoperoxidase method, except that the primary antibodies against HB-EGF and Ki67 were replaced by non-immune goat or mouse IgG (Miles, USA) at the same dilution as the specific antibodies. In the above-mentioned controls, the replacement of the specific primary antibodies with non-immune IgG resulted in a lack of positive immunostaining for HB-EGF and Ki67 respectively.

The cells immunostained for Ki67 were analysed and scored by two investigators in a blind fashion without knowledge of the experimental group. All of the stained nuclei were scored to be positive for Ki67. The mean percentage of Ki67-positive nuclei for each group of cultured leiomyoma cells and myometrial cells was determined by observing >1000 nuclei for each of the experimental samples and was used to evaluate the proliferating activity of the cells.

Western blot analysis
Proteins were extracted from cultured leiomyoma cells and myometrial cells as described previously (Shimomura et al., 1998Go). At the termination of cultures, cultured leiomyoma cells and myometrial cells were incubated at 4 °C for 20 min in the presence of lysis buffer consisting of 150 mmol/l NaCl, 2 mmol/l phenylmethylsulphonyl fluoride, 1% Nonidet P-40, 0.5% deoxycholate, 1 mg/l aprotinin, 0.1% sodium dodecylsulphate and 50 mmol/l Tris–HCl (pH 7.5). Cells were subsequently scraped off the plates. The lysates were centrifuged at 13 000 g for 3 min and the supernatants were collected. Protein contents in the supernatants were determined by the assay of Bradford (1976)Go. Each 60 µg aliquot of the proteins extracted from cultured cells was electrophoresed on a 4–12% NuPAGE Gel (NP0322Box; Invitrogen, USA) and transferred to polyvinylidene difluoride membrane. The bolts were exposed to a monoclonal antibody to PCNA and a polyclonal antibody to HER1 at the same dilution of 1:1000 in TBS–Tween (TBS-T) respectively. The antigen–antibody complex was detected with the secondary antibody using an ECL chemiluminescence detection system (Amersham Pharmacia Biotech). Membranes were visualized by exposure to X-OMAT film (Eastman Kodak Co., USA). The radioautograms were scanned and quantified with ChemiImager 4400 (Astec Co., Ltd, Japan). The experiments were repeated with at least three independent cultured specimens with similar results, and the reported results are representative.

TUNEL assay
ApopTag In Situ Apoptosis Detection kits (Intergen Co., USA) were used to identify apoptosis in cultured leiomyoma cells and myometrial cells. Cultured cells were fixed in 1% paraformaldehyde in PBS for 10 min and washed with PBS twice. The sections were fixed in pre-cooled ethanol:acetic acid (2:1) for 5 min at –20 °C and washed with PBS twice. After quenching endogenous peroxidase activity with 3% hydrogen peroxide in PBS, the sections were placed in equilibration buffer and then in TdT enzyme, followed by stop-wash buffer. After applying anti-digoxigenin peroxidase, peroxidase was detected with diaminobenzidine substrate working solution for 5 min. The sections were counterstained with 0.4% Methyl Green. The cells immunostained for apoptosis were analysed and scored by two investigators in a blind fashion without knowledge of the experimental group. All of the stained nuclei were scored as positive for apoptosis. The mean percentage of TUNEL-positive nuclei for each group of cultured myometrial cells and leiomyoma cells was determined by observing >1000 nuclei for each of the experimental samples and was used to evaluate the apoptotic cell death.

Statistical analysis
The data were expressed as the mean ± SD from four independent experiments. Statistical significance was evaluated using analysis of variance. Differences with P<0.05 were considered statistically significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Immunostaining for Ki67 in leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF
Leiomyoma cells and myometrial cells were cultured in serum-free conditions in the presence or absence of graded concentrations of HB-EGF for 24 and 48 h. Immunocytochemical examination demonstrated that immunoreactive Ki67 was localized to the nuclei of cultured leiomyoma cells and myometrial cells (Figure 1). In leiomyoma cells and myometrial cells treated with 10 ng/ml HB-EGF (Figure 1C, F, I, L) for 24 and 48 h, Ki67-positive nuclei were more abundant compared with those of untreated cultures (Figure 1A, D, G, J). However, there were no apparent differences in the Ki67 expression in leiomyoma cells and myometrial cells between cultures treated with 0.1 ng/ml HB-EGF for 24 and 48 h (Figure 1B, E, H, K) and untreated cultures (Figure 1A, D, G, J).



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Figure 1. Immunocytochemical staining for Ki67 in leiomyoma cells and myometrial cells cultured in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF). Leiomyoma cells cultured for 24 h without any treatment (A), cultured for 24 h in the presence of 0.1 ng/ml HB-EGF (B), cultured for 24 h with 10 ng/ml HB-EGF (C), cultured for 48 h without any treatment (D), cultured for 48 h with 0.1 ng/ml HB-EGF (E), cultured for 48 h with 10 ng/ml HB-EGF (F). Myometrial cells cultured for 24 h without any treatment (G), cultured for 24 h with 0.1 ng/ml HB-EGF (H), cultured for 24 h with 10 ng/ml HB-EGF (I), cultured for 48 h without any treatment (J), cultured for 48 h with 0.1 ng/ml HB-EGF (K), cultured for 48 h with 10 ng/ml HB-EGF (L). Bars = 50 µm.

 
The Ki67-positive rate in leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF
Figure 2 shows the mean percentage of Ki67-positive nuclei of leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF for 24 and 48 h. Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate (% control) in leiomyoma cells (upper panel) and myometrial cells (middle panel) compared with that in untreated cultures. However, treatment with 0.1 ng/ml HB-EGF did not affect the Ki67-positive rate (% control) in either leiomyoma cells (upper panel) or myometrial cells (middle panel) compared with that in untreated cultures.



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Figure 2. The Ki67-positive rate (% control) in leiomyoma cells and myometrial cells cultured in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF). The upper and middle panels show the mean percentage of Ki67-positive nuclei of leiomyoma cells and myometrial cells, respectively, cultured in the presence or absence of HB-EGF for 24 and 48 h. Treatment with HB-EGF at concentrations >1 ng/ml significantly increased the Ki67-positive rate in leiomyoma cells (upper panel) and myometrial cells (middle panel) compared with that in untreated cultures. The lower panel shows the comparison of the Ki67-positive rate between leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF for 24 and 48 h. The stimulatory effect of HB-EGF on the Ki67-positive rate was significantly greater in cultured myometrial cells compared with that in cultured leiomyoma cells. Data represent the mean ± SD of four determinations. *P<0.01, **P<0.05 versus untreated control cultures.

 
The lower panel shows the comparison of the Ki67-positive rate (% control) between leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF for 24 and 48 h. The stimulatory effect of HB-EGF on the Ki67-positive rate was significantly higher in cultured myometrial cells compared with that in cultured leiomyoma cells.

Effects of HB-EGF on PCNA expression in cultured leiomyoma cells and myometrial cells
Figure 3 shows PCNA expression in leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF after 24 h of culture, as assessed by western blot analysis. Western blot analysis revealed that cultured myometrial cells (upper panel) and leiomyoma cells (lower panel) contained immunoreactive PCNA protein with a molecular mass of ~36 kDa. Treatment with HB-EGF resulted in a dose-dependent increase in PCNA expression in both cells compared with untreated control cultures. A significant increase (P<0.01) in PCNA expression in cultured myometrial cells was noted by the treatment with HB-EGF at concentrations >1 ng/ml, whereas a significant increase in cultured leiomyoma cells was noted by the treatment with HB-EGF at concentrations >10 ng/ml.



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Figure 3. Proliferating cell nuclear antigen (PCNA) expression in leiomyoma cells and myometrial cells cultured after 24 h in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF), as assessed by western blot analysis. Western blot analysis revealed that cultured myometrial cells (upper panel) and leiomyoma cells (lower panel) contained immunoreactive PCNA protein with a molecular mass of ~36 kDa. PCNA expression in cultured myometrial cells was significantly increased in a dose-dependent manner when treated with HB-EGF compared with untreated control cultures (upper panel). By contrast, treatment with HB-EGF at concentrations >10 ng/ml significantly increased PCNA expression in cultured leiomyoma cells (lower panel). Experiments were repeated four times with similar results. Densitometric analysis was performed as described in Materials and methods. Data are presented as the fold increase over the control value and as the mean ± SD of four independent experiments. *P<0.01, **P<0.05 versus untreated control cultures.

 
Effects of HB-EGF on HER1 expression in cultured leiomyoma cells and myometrial cells
Figure 4 shows HER1 expression in leiomyoma cells and myometrial cells cultured for 24 h in the presence or absence of HB-EGF, as assessed by western blot analysis. Western blot analysis revealed that cultured leiomyoma cells and myometrial cells contained immunoreactive HER1 protein with a molecular mass of ~170 kDa. HER1 expression was significantly (P<0.01) higher in untreated myometrial cells than in untreated leiomyoma cells (upper panel). Treatment with HB-EGF significantly increased HER1 expression in both cell types. A significant increase (P<0.01) in HER1 expression in cultured myometrial cells was observed by the treatment with HB-EGF at concentrations >10 ng/ml (middle panel), whereas the significant increase (P<0.01) in HER1 expression in cultured leiomyoma cells was noted only by the treatment with HB-EGF at concentrations of 100 ng/ml (lower panel).



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Figure 4. Human epidermal growth factor receptor (HER1) expression in leiomyoma cells and myometrial cells cultured for 24 h in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF), as assessed by western blot analysis. Western blot analysis revealed that cultured leiomyoma cells and myometrial cells contained immunoreactive HER1 protein with a molecular mass of ~170 kDa (upper panel). HER1 expression was significantly higher in untreated myometrial cells than in untreated leiomyoma cells (upper panel). A significant increase in HER1 expression in cultured myometrial cells was observed by the treatment with HB-EGF at concentrations >10 ng/ml (middle panel), whereas the significant increase in HER1 expression in cultured leiomyoma cells was noted only by the treatment with HB-EGF at concentrations of 100 ng/ml (lower panel). Experiments were repeated four times with similar results. Densitometric analysis was performed as described in Materials and methods. Data are presented as the fold increase over the control value and as the mean ± SD of four independent experiments. MC = myometrial cells; LC = leiomyoma cells. *P<0.01 versus LC or untreated control cultures.

 
TUNEL assay in leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF
In leiomyoma cells and myometrial cells cultured in the presence of 10 ng/ml HB-EGF (Figure 5C, F, I, L) for 24 and 48 h, TUNEL-positive nuclei were less abundant compared with those in untreated cultures (Figure 5A, D, G, J). However, there were no differences in the abundance of TUNEL-positive nuclei in leiomyoma cells and myometrial cells between cultures treated with 0.1 ng/ml HB-EGF for 24 and 48 h (Figure 5B, E, H, K) and untreated cultures (Figure 5A, D, G, J).



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Figure 5. TUNEL assay in leiomyoma cells and myometrial cells cultured in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF). Leiomyoma cells cultured for 24 h without any treatment (A), cultured for 24 h with 0.1 ng/ml HB-EGF (B), cultured for 24 h with 10 ng/ml HB-EGF (C), cultured for 48 h without any treatment (D), cultured for 48 h with 0.1 ng/ml HB-EGF (E), cultured for 48 h with 10 ng/ml HB-EGF (F). Myometrial cells cultured for 24 h without any treatment (G), cultured for 24 h with 0.1 ng/ml HB-EGF (H), cultured for 24 h with 10 ng/ml HB-EGF (I), cultured for 48 h without any treatment (J), cultured for 48 h with 0.1 ng/ml HB-EGF (K), cultured for 48 h with 10 ng/ml HB-EGF (L). Bars = 50 µm.

 
The TUNEL-positive rate in leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF
The upper and middle panels of Figure 6 show the mean percentage of TUNEL-positive nuclei in leiomyoma cells and myometrial cells, respectively, cultured in the presence or absence of HB-EGF for 24 and 48 h. Treatment with HB-EGF at concentrations >1 ng/ml significantly decreased the TUNEL-positive rate (% control) in leiomyoma cells (upper panel) and myometrial cells (middle panel) compared with that in untreated cultures. However, treatment with 0.1 ng/ml HB-EGF did not affect the TUNEL-positive rate (% control) in either leiomyoma cells (upper panel) or myometrial cells (middle panel) compared with that in untreated cultures. The lower panel shows the comparison of the TUNEL-positive rate (% control) between leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF for 24 and 48 h. No significant differences in the TUNEL-positive rate were noted between leiomyoma cells and myometrial cells cultured in the presence of HB-EGF.



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Figure 6. TUNEL-positive rate (% control) in leiomyoma cells and myometrial cells cultured in the presence or absence of heparin-binding epidermal growth factor-like growth factor (HB-EGF). The upper and middle panels show TUNEL-positive rate in leiomyoma cells and myometrial cells, respectively, cultured in the presence or absence of HB-EGF for 24 and 48 h. Treatment with HB-EGF at concentrations >1 ng/ml significantly decreased the TUNEL-positive rate in leiomyoma cells (upper panel) and myometrial cells (middle panel) compared with that in untreated cultures. The lower panel shows the comparison of the TUNEL-positive rate between leiomyoma cells and myometrial cells cultured in the presence or absence of HB-EGF for 24 and 48 h. No significant differences in the TUNEL-positive rate were noted between leiomyoma cells and myometrial cells treated with HB-EGF. Data represent the mean ± SD of four determinations. *P<0.05, **P < 0.01 versus untreated control cultures.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In the present study, we have demonstrated that treatment with HB-EGF significantly increased the Ki67-positive rate of leiomyoma cells and myometrial cells in a dose-dependent manner at 24 and 48 h of treatment. Treatment with HB-EGF also significantly increased PCNA expression in those cells as early as 24 h of culture. This was consistent with results obtained with Ki67 immunostaining. The Ki67 nuclear antigen is expressed in the proliferating cells during the late G1, S, M and G2 phases of cell cycle (McCormick et al., 1993Go; Schluter et al., 1993Go). PCNA, a co-factor of DNA polymerase {delta}, has been demonstrated to play an essential role in DNA replication and repair (Kelman, 1997Go). The fact that HB-EGF increased immunocytological Ki67 labelling and PCNA expression in cultured leiomyoma cells and myometrial cells indicates that HB-EGF exerts stimulatory effects on the proliferative potential in those cells. Interestingly, the effects of HB-EGF on the Ki67-positive rate and PCNA expression were greater in myometrial cells than in leiomyoma cells. Our results are in accord with the reports showing that HB-EGF is a potent mitogen for bovine aortic smooth muscle cells (Higashiyama et al., 1991Go) and rat vascular smooth muscle cells (Fukuda et al., 1997Go). HB-EGF-induced DNA synthesis in rat vascular smooth muscle cells has recently been reported to require extracellular signal-regulating kinase (Erk) and phosphatidylinositol 3-kinase pathways through HER1 (Reynolds et al., 2002Go). However, the signalling pathways by which HB-EGF regulates the proliferation of leiomyoma cells and myometrial cells remain to be elucidated.

The expression of HB-EGF in the uterus has been shown to be regulated by sex-steroid hormones (Wang et al., 1994Go; Zhang et al., 1994Go). Zhang et al. (1994)Go reported that in ovariectomized rat uterus, progesterone increased HB-EGF mRNA levels in uterine stromal cells, but repressed HB-EGF expression in uterine epithelial cells, whereas 17{beta}-estradiol treatment enhanced HB-EGF expression in epithelial cells, but no effect on HB-EGF mRNA levels in stromal cells. Progesterone treatment followed by estradiol stimulated HB-EGF expression in stromal cells, but repressed expression in epithelial cells. Moreover, it has been reported that the expression of HB-EGF in the ovary (Kennedy et al., 1993Go) and endometrium (Das et al., 1994Go; Tazuke and Giudice, 1996Go) is more abundant during the secretory phase of the menstrual cycle than during the proliferative phase of the menstrual cycle. Treatment with pure estrogen receptor antagonists such as ICI 182,780 and ICI 164,384 has been reported to prevent estradiol-induced HB-EGF liberation in cultured MCF-7 cells (Razandi et al., 2002Go) and inhibit estradiol-induced increases in HB-EGF mRNA in rat uterine epithelial cells (Zhang et al., 1998Go). These results suggest that the blockade of estrogen may suppress the effect of HB-EGF. However, the precise mechanism responsible for the regulation of HB-EGF in leiomyoma cells and myometrial cells remains unknown.

Several studies have demonstrated the presence of erbB1 mRNA (Yeh et al., 1991Go) and immunoreactive HER1 protein (Leone et al., 1991Go; Shimomura et al., 1998Go; Dixon et al., 2000Go) in leiomyoma cells and myometrial cells. Scatchard analysis demonstrated that EGF receptor binding sites per cell were significantly lower in leiomyoma cells than in myometrial cells (Fayed et al., 1989Go). To clarify the mechanism by which myometrial cells responded better to the proliferation stimulatory effect of HB-EGF compared with leiomyoma cells, we investigated the effect of HB-EGF on HER1 expression in those cells. Western blot analysis demonstrated that HER1 expression was more abundant in untreated myometrial cells than in untreated leiomyoma cells, and that treatment with HB-EGF augmented HER1 expression in cultured leiomyoma cells and myometrial cells. However, the higher concentrations of HB-EGF were required to attain the significant increase in HER1 in cultured leiomyoma cells compared with cultured myometrial cells, reinforcing the notion that cultured myometrial cells respond better to HB-EGF than cultured leiomyoma cells. It seems likely that HB-EGF acts to promote cell proliferation by up-regulating HER1 expression in those cells. The difference in responsiveness to the proliferation stimulatory effect of HB-EGF between cultured leiomyoma cells and myometrial cells may be due to the difference in the levels of HER1 expression and intracellular signal pathways after binding to HB-EGF. It seems reasonable to speculate that through the interaction with more abundant and enhanced activation of HER1, cultured myometrial cells can respond better to HB-EGF than cultured leiomyoma cells.

Numerous studies have demonstrated that HB-EGF plays an important role in the regulation of cell survival in a variety of normal cells and cancer cells (Miyoshi et al., 1997Go; Takemura et al., 1997Go; Zushi et al., 1997Go; Horikawa et al., 1999Go; Iwamoto et al., 1999Go; Nguyen et al., 2000Go; Fang et al., 2001Go; Michalsky et al., 2001Go; Farkas and Krieglstein, 2002Go). HB-EGF has been shown to promote survival of dopaminergic neurons through activation of the mitogen-activated protein kinase (MAPK) and Akt signalling pathways (Farkas and Krieglstein, 2002Go). In the present study, treatment with HB-EGF significantly inhibited apoptosis in cultured leiomyoma cells and myometrial cells. This suggests that cross-talk between HB-EGF and HER1 may contribute to the suppression of apoptosis in those cells. However, the reason for no significant difference in the apoptosis-inhibiting effect of HB-EGF between leiomyoma cells and myometrial cells remains unknown.

In conclusion, we have demonstrated that HB-EGF stimulates the growth of cultured leiomyoma cells and myometrial cells by up-regulating PCNA and inhibits apoptosis of these cells. In this context, HER1 expression was higher in untreated myometrial cells than in untreated leiomyoma cells, and treatment with HB-EGF up-regulated HER1 expression in those cells. Cultured myometrial cells may have responded better to the proliferation stimulatory effects of HB-EGF due to the greater abundance and enhanced activation of HER1 compared with cultured leiomyoma cells. It is therefore likely that the role of HB-EGF may be more vital in myometrial growth than in leiomyoma growth. Further studies will be needed to elucidate the mechanisms by which HB-EGF regulates the growth and apoptosis of myometrial cells and leiomyoma cells through the interaction with HER.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
This work was supported in part by Grants in Aid for Scientific Research 1437053 from the Japanese Ministry of Education, Science and Culture and by the Ogyaa-Donation Foundation of Japan Association of Obstetricians and Gynecologists.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on June 22, 2004; resubmitted on January 18, 2005; accepted on February 14, 2005.





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