1 Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo, 113-8655, 2 Cell Signals Inc., 505 Leading Venture Plaza, 75-1 Ono Tsurumi-ku, Yokohama 230-0046 and 3 Department of Health Science, Faculty of Psycological and Physical Sciences, Aichi Gakuin University, 12 Araike-cho, Nisshin, Aichi 470-0915, Japan
4 To whom correspondence should be addressed: Email: yutakaos-tky{at}umin.ac.jp
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Abstract |
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Key words: adhesions/endometriosis/midkine/peritoneal fluid/cell proliferation
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Introduction |
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Midkine (MK) is a basic, low molecular weight nonglycosylated protein, composed of two domains held by disulfide bridges. MK belongs to a member of the heparin-binding growth factor family and has been identified as the product of a retinoic acid-responsive gene. MK plays important roles in development and is strongly expressed during midgestation. Although its expression is restricted to certain tissues in the adult, it is strongly induced during oncogenesis, inflammation and tissue repair. MK has pleiotropic activities, such as cell proliferation, cell migration, angiogenesis and fibrinolysis. Its receptor is considered to be a molecular complex containing receptor-type tyrosine phosphatase z (PTPz), low density lipoprotein receptor-related protein (LRP), anaplastic leukemia kinase (ALK) and syndecans (Muramatsu, 2002).
MK has been detected in the endometrium, the levels being high in women with endometriosis as compared to those in women without the disease (Chung et al., 2002). In view of high levels of MK in a number of malignant tumors, suggesting its involvement in cancer development (Tsutsui et al., 1993
; Garver et al., 1994
; Aridome et al., 1995
; Nakagawara et al., 1995
; O'Brien et al., 1996
; Koide et al., 1999
; Konishi et al., 1999
; Kato et al., 2000
), increased MK in the endometrium of women with endometriosis is speculated to subserve proliferation of refluxed endometrial cells and to promote the formation of endometriotic foci on the peritoneum in an autocrine/paracrine fashion. In addition, a recent study demonstrating that MK plays key roles in intraperitoneal adhesions imply its possible relevance to endometriosis-associated intraperitoneal adhesion (Inoh et al., 2004).
In the present study, to address the possible implication of MK in endometriosis, we first evaluated whether MK has a proliferative effect on cultured endometriotic stromal cells (ESC). Secondly, considering the importance of peritoneal environment for the development of endometriosis, we examined the concentrations of MK in PF of women with or without endometriosis, and its possible sources.
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Materials and methods |
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Endometriotic tissues were collected from cyst walls of ovarian endometriomas of three patients without GnRHa therapy. Likewise, normal peritonea that did not include endometriotic lesions were collected under laparoscopy. The tissues for RNA extraction were snap-frozen in liquid nitrogen, and stored at 80 °C until assay.
Twenty follicular fluids were collected from preovulatory follicles of 20 patients mostly with r-ASRM score 0.5 by needle aspiration under laparoscopy.
Isolation, purification and culture of ESC
Primary cultures of ESC were prepared from the obtained endometriotic tissues according to the method described by Ryan et al. (1994) with minor modifications. Endometriotic tissue was dissected free from underlying parenchyma, minced into small pieces, incubated in Dulbecco's modified Eagle's medium (DMEM)/F12 with 0.25% type I collagenase (Sigma, St Louis, MO) and SU/ml deoxyribonuclease I (Takara, Tokyo, Japan) for 12 h at 37 °C, and separated using serial filtration. Debris and some epithelial glands were removed with a 100 µm and a 70-µm nylon cell straine (Becton Dickinson, Lincoln Park, NJ), and some epithelial glands were removed with a 70 µm nylon cell strainer (Becton Dickinson). Stromal cells remaining in the filtrate were collected by centrifugation, resuspended in DMEM/F12, and plated onto 100-mm dishes and allowed to adhere at 37 °C for 30 min, after which nonadhering epithelial cells and blood cells were removed with PBS rinses. The cells were cultured in DMEM/F12 reconstituted with 10% charcoal-stripped fetal bovine serum (HyClone, Logan, UT) and antibiotics (Sigma). When the cells become confluent, they were dissociated with 0.25% trypsin, harvested by centrifugation, replated in Falcon 96-multiwell plates (Becton Dickinson) at 1 x 104 cells/well, and kept at 37 °C in a humidified 5% CO2/95% air environment. After 24 h, purification of the stromal cell population was confirmed by immunocytochemical staining for the following antibodies (Dako, Kyoto, Japan): vimentin (stromal cells), cytokeratin (epithelial cells) and CD45 (monocytes and other leukocytes). The purity of the stromal cells was >98%, as judged by positive cellular staining for vimentin and negative cellular staining for cytokeratin and CD45.
Cell proliferation assay
The cell proliferation assay was performed as we have reported previously (Tang et al., 2002). The effect of MK on the proliferation of ESC was examined by measuring 5-bromo-2'-deoxyuridine (BrdU) incorporation into DNA using the Biotrak cell proliferation enzyme-linked immunosorbent assay (ELISA) system (Amersham Biosciences) according to the manufacturer's instructions. Briefly, ESC were seeded into Falcon 96-multiwell plates at a density of 1 x 104 cells per well in 100 µl of the culture medium with MK at different concentrations (0, 10, 100 and 1000 ng/ml) or with 20% PF from patients of endometriosis (r-ASRM score
6) (Braun et al., 2002
). After 24 h, 100 µl of BrdU solution was added and incubated at 37 °C for an additional 2 h. After removing the culture medium, the cells were fixed and the DNA denatured by the addition of 200 µl/well of fixative. The peroxidase-labeled anti-BrdU bound to the BrdU incorporated in the newly synthesized cellular DNA. The immune complexes were detected by the subsequent substrate reaction, and the resultant color was read at 450 nm in the DigiScan Microplate Reader (ASYS Hitech GmbH, Eugendorf, Austria).
Measurement of MK
Concentrations of MK in PF were determined using an ELISA kindly provided by Cell Signals (Yokohama, Japan). The sensitivity limit of this assay was 0.1 ng/ml per sample.
RNA extraction and RTPCR of MK mRNA
Total RNA was extracted from peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RTPCR was performed using Rever Tra Dash (Toyobo, Tokyo, Japan). One microgram of total RNA was reverse-transcribed in a total volume of 20 µl, and complementary DNA (cDNA) was amplified using oligonucleotide primers based on the human MK sequence. The human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers (Toyobo) were used to ensure RNA quality and amounts. MK primers (sense, 5'-CCTGCAACTGGAAGAAGGAG-3'; antisense, 5'-AGCAGACAGAAGGCACTGGT-3') were chosen to amplify a 320 bp fragment. PCR conditions for the amplifications of MK and GAPDH were 30 cycles at 98 °C for 10 s, 60 °C for 2 s, and 74 °C for 20 s. PCR products were analysed by agarose gel electrophoresis with ethidium bromide. Each PCR product was purified with a QIAEX II gel extraction kit (Qiagen), and their identities were confirmed using an ABI PRISMTM 310 genetic analyzer (Applied Biosystems, Foster City, CA).
Statistical analysis
Analysis of variance was used to compare the BrdU incorporation of the cultured cells. The data of MK in PF were described as median and interquartile range (IQR). MannWhitney test was used to compare concentrations of MK in PF. P<0.05 was considered significant.
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Results |
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As demonstrated in Figure 3, the expression of MK mRNA was detected as a clear band at 320 bp in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues.
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Discussion |
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MK has pleiotropic functions not only for promoting cell proliferation, as demonstrated in the endometriotic cells in the present study, but also for inducing angiogenesis and inflammation (Takada et al., 1997; Horiba et al., 2000
). All these functions are suggested to be important for developing tumors and tumor-like lesions such as endometriosis. Indeed, MK expression has been shown to increase in a number of malignant tumors compared to that in the adjacent non-cancerous tissue (Aridome et al., 1995
; Koide et al., 1999
; Konishi et al., 1999
), suggesting its relevance in tumor formation. Interestingly, eutopic endometrial cells of women with endometriosis expressed increased levels of MK as compared to those of women without endometriosis (Chung et al., 2002
). In the light of the proliferative effect of MK on ESC, the increased levels of MK in eutopic endometrial cells may contribute, in part, to the formation of endometriotic foci when they are refluxed into the peritoneal cavity.
Consistent with the notion that peritoneal environment is important for the development of endometriosis, the concentration of MK in PF of women with advanced endometriosis was increased. As shown in this study, in addition to endometriotic tissues, peritoneal bone marrow-derived cells and peritoneal tissues produce MK. Given that locally produced MK stimulates the proliferation of ESC, it may be plausible that the concentrations of MK in PF are relatively low, compared to those effective in vitro, possibly due to dilution. These findings also imply that MK may stimulate the growth of endometriotic lesions in an autocrine and paracrine fashion, although ectopic endometriotic lesions express lower levels of MK than eutopic endometrium (Chung et al., 2002).
A recent report has shown that MK stimulates intraperitoneal adhesion in a mouse model of peritoneal injury (Inoh et al., 2004). The present study demonstrated that the concentrations of MK in women with adnexal adhesions are increased as compared with those without adhesions. In view of the propensity of endometriosis to form peritoneal adhesions along the progress of the disease, MK might also contribute to the adhesion formation.
The sources of MK in PF appear to be widely distributed. Our RTPCR analysis demonstrated that MK is expressed in peritoneal bone marrow-derived cells, peritoneum and endometriotic tissues. In addition, MK in follicular fluid discharged at ovulation could constitute a part of MK in PF. Similar to bovine follicular fluid which contains large amounts of MK (Ohyama et al., 1994), we found high concentrations of MK in human ovarian follicles, over 200-fold higher than those in PF. The high concentration of MK in follicular fluid may partly explain the finding that MK in PF was increased in the luteal phase as compared to that in the follicular phase. On the other hand, it might be partially due to the lack of follicular fluid influx to PF that the difference of the MK concentrations between advanced endometriosis and no/minimal endometriosis was more evident in the follicular phase than in the luteal phase.
A remarkable finding in this study is that MK levels in the women undergoing GnRHa treatment were significantly lower as compared to those in the women with advanced endometriosis and those with minimal or no endometriosis. It has been reported that estradiol (E2) induced MK mRNA expression in endometrial epithelial cells (Zhang et al., 1995). The several estrogen responsive element half-palindromic motifs (Kato et al., 1992
) residing in the promoter region of the MK gene (Uehara et al., 1992
) have been suggested to cause this effect of E2 (Zhang et al., 1995
). Hypoestrogenic status induced by GnRHa treatment may suppress gene transcription of MK in various cells. In addition, anovulation caused by GnRHa treatment could contribute to the suppression of MK levels in PF, hindering the flux of follicular fluid into the peritoneal cavity.
MK has been shown to proliferate in several cells, including fibroblasts, tumor cells and keratinocytes (Muramatsu and Muramatsu, 1991; Muramatsu et al., 1993
; Inazumi et al., 1997
). The signaling pathways mediating the MK effects are only partially revealed at present. While receptor complexes containing PTPz, LRP, ALK and syndecans are suggested to be at work for MK functions (Muramatsu, 2002
; Sakaguchi et al., 2003
; Deepa et al., 2004
), LRP and syndecan-1 have been shown to be present in the endometrium (Sayegh et al., 1995
; Inki, 1997
; Foca et al., 2000
). In view of these findings, it may be interesting to explore the therapeutic potential of MK inhibitors for endometriosis. In mouse rectal carcinoma cells, antisense oligonucleotides to MK have been shown to suppress tumor formation (Takei et al., 2001
).
In summary, the present study demonstrated that MK stimulates the proliferation of ESC and that MK levels are increased in the PF of women with advanced endometriosis, suggesting that MK may play a role in the development of endometriosis.
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Acknowledgements |
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References |
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Submitted on May 19, 2004; resubmitted on August 23, 2004; accepted on December 7, 2004.